Quantitative changes in the phosphoproteome of human cell lines following infection by the human immunodeficiency virus (HIV) were analysed by hybrid Orbitrap-based mass spectrometry. Global identification and quantification of changes in human phosphoproteome in response to HIV was performed using stable isotope labeled (SILAC) Jurkat cells and HILIC/TiO2 fractionation and enrichments using the new Orbitrap FusionTM TribridTM mass spectrometer and EASY-ETDTM source. More phosphopeptides were identified using a combination of ETD and CID fragmentation in a single run (via novel data dependent decision tree with dynamic scan management) than when using CID fragmentation alone which permitted precise localization of phosphorylation more frequently.