Kinase and phosphatase overexpression drives tumorigenesis and drug resistance. We previously developed a mass-cytometry-based single-cell proteomics approach that enables quantitative assessment of overexpression effects on cell signaling. Here, we applied this approach in a human kinome- and phosphatome-wide study to assess how 649 individually overexpressed proteins modulated cancer-related signaling in HEK293T cells in an abundance-dependent manner. Based on these data, we expanded the functional classification of human kinases and phosphatases and showed that the overexpression effects include non-catalytic roles. We detected 208 previously unreported signaling relationships. The signaling dynamics analysis indicated that the overexpression of ERK-specific phosphatases sustains proliferative signaling. This suggests a phosphatase-driven mechanism of cancer progression. Moreover, our analysis revealed a drug-resistant mechanism through which overexpression of tyrosine kinases, including SRC, FES, YES1, and BLK, induced MEK-independent ERK activation in melanoma A375 cells. These proteins could predict drug sensitivity to BRAF-MEK concurrent inhibition in cells carrying BRAF mutations. Display omitted •Human kinome- and phosphatome-wide screen of overexpression-altered signaling network•Classification of the kinome and phosphatome based on abundance-dependent effects•Phosphatase overexpression sustains MAPK-ERK signaling by halting negative feedback•SRC, FES, YES1, and BLK lead to drug resistance in BRAF-MEK combined inhibition Lun et al. perform mass-cytometry-based single-cell analysis to study how the expression levels of 649 kinases and phosphatases affect the intracellular signaling network. They expand the functional classification of the kinome and phosphatome based on abundance-dependent effects and identify novel signaling mechanisms underlying overexpression-driven diseases and drug resistance.