Weigh 5-10 flies in a pre-weighed eppendorf tube.    Freeze the flies in liquid nitrogen. This prevents proteases from degrading your proteins and protects the protein conformational state of your enzymes (if needing to assay their activity, use an appropriate buffer).  Transfer them quickly to ice and add RIPA buffer (10ul/1mg of flies) with protease inhibitors (use a mini-cocktail from a company like Roche or ThermoFisher).   Using a hand-held homogenizer, homogenize the flies in ice 6X for 2-3 min each.  Leave it on ice for 10 min to extract as many proteins as possible.   Add 2X SDS loading dye with DTT at a 1:1 ratio to prepare your proteins for running them in the gel (coats them with a negative charge and cleaves the sulfhydril bonds to linearize them).  Boil at 95oC for 5 min (change the temperature and time depending on the hydrophobicity of your protein of interest, i.e. 65oC for 30 min for some membrane proteins).   Good luck! Let me know if you have any problems.      

Alessia, do you include the whole head in your PBS preparation?  How fast do you spin it and for how long?  You don't use a homogenizer? Thanks!

Looking for an effective protocol if the sample volume is less, say one or two flies, and for further technique like I Trac  

You can try the attached protocol. It may be useful