Competitive proteome profiling is a powerful approach for the identification of targets of small molecules. This approach usually employs an inhibitor-derived probe or a cysteine-reactive probe such as an IA-alkyne in a comparison between inhibitor-treated and untreated samples, thus enabling distinction between genuine targets and nonspecific labeling. We have developed an active probe derived from an EGFR inhibitor, afatinib, and a cysteine reactive probe, an alkyne-containing α,β-unsaturated amide, to compare their characterization of cellular targets. In both approaches, myosin heavy chain 9 (MYH9) was identified as an off-target. Subsequent functional validation experiments suggested that MYH9 might be involved in the function of afatinib.© 2022 Wiley-VCH GmbH.