In conventional data-dependent acquisition (DDA) mode used for phosphoproteome analysis by mass spectrometry, a considerable number of phosphosites are localized ambiguously. We found that data-independent acquisition (DIA) method could localize phosphosite accurately and confidently due to its acquisition nature of repeated and unbiased mode. Here, we present a robust and promising DIA workflow for modification sites determination. Through dedicated collection and analyses of site-determining ions following their chromatographic profilings, phosphosites could be assigned or rectified unambiguously.

Data-Independent Acquisition Mass Spectrometry to Localize Phosphosites