Double-stranded RNA (dsRNA) binding proteins play key roles in numerous biological activities in cells, including innate immunity, RNA interference, and mRNA transport. Here, we established an analytical strategy to characterize dsRNA binding proteins from naive cell lysate by segmented proteome integral solubilityalteration assay combined with label free quantitative mass spectrometry. In this approach, we identified 218 dsRNA binding proteins from naive cell lysate of Hela cell. The data confirmed 18 known dsRBPs and identified 200 novel dsRBPs which have never been reported before.