It is a common misconception that taking damage while stealthed will break stealth: it does not. Even if revealed by True Sight, taking damage while stealthed will not remove stealth.
Taking damage will only delay you entering stealth, during the 1.25 seconds after activating Ambush.
Stealth will only be broken if the stealthed champion attacks or activates an ability.
Stealthed champions can use summoner spells without breaking stealth, such as Ignite and Smite. The ability to cast Smite without breaking stealth makes Twitch, Evelynn and to some extent Teemo strong jungle invaders or counter-invaders.
Consuming items will not break stealth (such as placing a Sight Ward or using a Health Potion). However, activating items will break stealth (such as Executioner's Calling and Wriggle's Lantern.
After Ambush has been activated, Twitch can activate Zhonya's Hourglass and he will be stealthed after the 2 seconds. This allows Twitch to avoid his enemies' attempt to delay him entering stealth.
Twitch can also cast Recall and Teleport during the 1.25 second delay to become stealthed during the channel. This allows Twitch to safely recall or appear at his teleport destination invisible.
Twitch can still be damaged while invisible, which will interrupt recall.
In Dominion, Twitch can click to capture points and relics during the delay to become stealthed. However, as enemy and neutral capture points, and the Storm Relic provide true sight, you will be revealed. This tactic merely allows you to escape without delay, rather than backdoor invisibly.
The true damage (damage before armor) is calculated against the original target, or the first enemy hit if the target avoids the shot.
If it would critically strike, it critically strikes everyone: the dice isn't rolled per enemy.
If the attack were to be blocked by effects such as Aegis Protection, Counter Strike and Riposte 0 damage will be dealt to all enemies. This is the case even if it visually hits other targets en route.
After the true damage has been calculated, damage mitigation is then applied per enemy. As such, targeting the tank (which may be advantageous for lining the skill shot) will not affect the damage dealt to all enemies.
This includes Aegis Protection, Counter Strike and Riposte. If said champions are not the original attack target, blocking the damage will only mitigate the damage against themselves.
If Twitch is blinded, Spray and Pray will miss and still consume a stack.
Spray And Pray will pass through any structures and terrain if it has targeted the designated enemy.
Spray and Pray often misses running enemies since Twitch will just aim after the current position of the enemy and shoot in a straight line.
Ambush will be in the category of "Stealth" and not "Invisiblity" (Upcoming Stealth changes).
Spray and Pray gives you longer range than towers, which means you can strike towers without them being able to strike back.
If Twitch dies stealthed, his corpse will be stealthed too. If the stealth timer runs out while dead, his corpse will reveal and laugh. If not, he will be stealthed after respawning.

Ambush

Active): After 1.25 seconds, Twitch becomes stealthed. If Twitch attacks or casts a spell while stealthed, he loses stealth but gains bonus attack speed. This attack speed bonus has duration equal to double the amount of time Twitch was stealthed before attacking, to a maximum duration of 10 seconds.
Cost: 60 mana
Cooldown: 11 seconds

Debilltating Poison

(Active): Slows nearby enemy champions by 30%, plus 6% per stack of Deadly Venom applied to them. Adding more Deadly Venom stacks after the slow is applied will further reduce their speed.
Cost: 80 mana
Cooldown: 10 seconds
Radius: 1200

Expunge

(Active): Deals magic damage to each nearby poisoned enemy, plus additional damage per stack of Deadly Venom applied to them. All stacks of Deadly Venom are removed from the targets.
Cost: 90 mana
Cooldown: 8 seconds
Radius: 1200

Spray and Pray

Active): Twitch's basic attacks become piercing skill shots that deal 100% of his attack damage and apply on-hit effects to all enemies in a line. If the attack would critically strike, the damage is multiplied against all affected enemies. This ability lasts for a maximum number of attacks or after 12 seconds. While active, Twitch is granted additional attack damage and has increased attack range.
Cost: 150 mana
Attack Range: 1200