In daily practice, different types of biomolecules are usually extracted for large-scale "omics" analysis with tailored protocols. However, when sample material is limited, an allin- one strategy is preferable. Although lysis of cells and tissues with urea is widely used for phosphoproteomic applications, DNA, RNA, and proteins can be simultaneously extracted from small samples using acid guanidinium thiocyanate-phenol-chloroform (AGPC). Use ofAGPC for mass spectrometry-based phosphoproteomics was reported but has not yet been thoroughly evaluated against a classical phosphoproteomic protocol. Herewecompared urea- with AGPC-based protein extraction, profiling phosphorylations in the DNA damage response pathway after ionizing irradiation of U2OS cells as proof of principle. On average we identified circa 9000 phosphosites per sample with both extraction methods. Moreover, we observed high similarity of phosphosite characteristics (e.g., 94% shared class 1 identifications) and deduced kinase activities (e.g., ATM, ATR, CHEK1/2, PRKDC). We furthermore extended our comparison to murine and human tissue samples yielding similar and highly correlated results for both extraction protocols. AGPC-based sample extraction can thus replace common cell lysates for phosphoproteomic workflows andmay thus be an attractive way to obtain input material for multiple omics workflows, yielding several data types from a single sample.