Protecting your brother Assassins can be difficult
A good offense is the best defense
The key to mastering the simple tower defense minigame is in the placement of your ally Assassins.
Start always by placing a few Assassin Leaders on rooftops, these generate Morale which is used to bring further units into the fray on your side. They also unlock the rooftop for placement of more units, and defend your ranged troops from Templars that get too close.
Also, place a couple of Crossbowmen early on, preferably around the middle of the street. This strikes the best balance of them being in range to attack the invading Templar force sooner, but still being far enough back that none will slip through.
Your next objective is to get as many Riflemen as possible onto the rooftops, ideally you want to aim for at least two on every rooftop before the final wave. This, along with liberal application of barricades the whole way along the street will be necessary to stall the siege engines employed by the Templars long enough for your units to take care of them. Riflemen fire slightly slower than the Crossbowmen, but deal armour-piercing damage, making them a lot more capable of bringing down the tough final wave enemies than any other unit, even the unlockable Bomb throwers or the Greek Fire Barricade.
Ideally you want to have a mixture of units, but concentrate mostly on having your rooftops lined with Riflemen, and basic level 1 Barricades inthe street. A good trick to remember is that when the Barricade is about to be destroyed, you can upgrade it to restore it’s health rather than simply increasing the durability between waves.
Caltrop Bomber Assassins will slow waves of enemies down, and so are best placed near the end of the street from which the Templars approach.
On later Sequences, the Templars will deploy rooftop units which will attempt to wipe out your vulnerable rooftop ranged attackers. Your role as Ezio is to use your Wrist Gun to pick them off as you would use it normally. It can also be used to get your allies to focus their fire on a particular group (any Templar ranged attackers should be prioritised, they can whittle down your numbers very quickly).
Also, L2/LT can be used to deploy a single cannon shot, which will decimate most of a wave of Templars if well placed. Holding it in will charge a 3 shot barrage. However, using the cannon will greatly reduce your score and the rewards that a high score entails (rare bomb ingredients, experience for your Assassins and some money) and prevent you from getting the Iron Curtain trophy.
The final wave will prove to be the most difficult, don’t be afraid to sell your frontal units to buy more for nearer the Den of the bulk of the enemy force gets through. Placing barricades in every space you can and upgrading them to counteract the damage should buy your Assassins enough time to slowly bring down the powerful siege engines. Make sure to keep firing at it yourself to keep your units focused on bringing it down.
Even if you fail - it’s not the end of the world. You can recapture the Den through more subtle methods, wherein you assassinate the Templar captain as normal and light the signal.