Mass spectrometry-based phosphoproteomics of tumor tissue or cell line lysates provides insight in aberrantly activated signaling pathways and potential drug targets. For improved understanding of the individual patient’s tumor biology, analysis of the phosphoproteome should be feasible and reproducible using tumor needle biopsies. We hereto scaled down a pTyr-phosphopeptide enrichment protocol to biopsy-level protein input and show its performance using colorectal cancer (CRC) cell line and needle biopsies from patients. In this study, the feasibility of label-free pTyr-phosphoproteomics at the biopsy level is demonstrated. Unsupervised cluster analysis shows that this approach can identify patient-specific profiles, which will improve our understanding of individual tumor biology and may enable future pTyr-phosphoproteomics-based TKI treatment selection.