The small-molecule drug NT157 has demonstrated promising efficacy in preclinical models of a number of different cancer types, reflecting activity against both cancer cells and the tumor microenvironment. Two known mechanisms of action are degradation of insulin receptor substrates (IRS)-1/2 and reduced Stat3 activation, although it is possible that others exist. To interrogate the effects of this drug on cell signaling pathways in an unbiased manner, we have undertaken mass spectrometry–based global tyrosine phosphorylation profiling of NT157-trea-ted A375 melanoma cells. Bioinformatic analysis of the resulting dataset resolved 5 different clusters of tyrosine-phosphorylated peptides that differed in the directionality and timing of response to drug treatment over time. The receptor tyrosine kinase AXL exhibited a rapid decrease in phosphorylation in response to drug treatment, followed by proteasome-dependent degradation, identifying an additional potential target for NT157 action. However, NT157 treatment also resulted in increased activation of p38 MAPK a and g, as well as the JNKs and specific Src family kinases. Importantly, cotreatment with the p38 MAPK inhibitor SB203580 attenuated the antiproliferative effect of NT157, while synergistic inhibition of cell proliferation was observed when NT157 was combined with a Src inhibitor. These findings provide novel insights into NT157 action on cancer cells and highlight how globally profiling the impact of a specific drug on cellular signaling networks can identify effective combination treatments.