Biochemical studies of cellular processes involving polyubiquitin have gained increasing attention. More tools are needed to identify ubiquitin (Ub)-binding proteins.W er eport diazirine-based photoaffinity probes that can capture Ubbinding proteins in cell lysates,a nd show that diazirines are preferable to aryl azides as the photo-crosslinking group,since they decrease non-selective capture.P hotoaffinity probes containing at least two Ub units were required to effectively capture Ub-binding proteins.D ifferent capture selectivity was observed for probes containing diubiquitin moieties with different types of linkages,t hus indicating the potential to develop linkage-dependent probes for selectively profiling Ubbinding proteins under various cellular conditions.