In this study we compared the phosphoproteome of control and cold acclimated Drosophila melanogaster in order to unravel the cold acclimation regulation mechanisms. For this prurpose we enriched phosphopeptides from each sample using a sequential elution from IMAC strategy (SIMAC) and analyzed them with a LTQ-OrbitrapXL mass spectrometer using a combination of different fragmentation strategies (classical data dependant analysis using CID, neutral loss scanning and multi-stage activation) in order to improve the number of identified phosphopeptides.