ALDH1A family enzymes (ALDH1A1, ALDH1A2, and ALDH1A3) catalyze retinoic acid synthesis, and their dysregulation is linked to disease. Selective inhibitors of these enzymes have been tested in drug discovery programs and one such compound, WIN18,446, was found to irreversibly inhibit ALDH1A2. WIN18,446 is a reversible male contraceptive in humans and animals. The inhibition of spermatogenesis by WIN18,446 is thought to result from inhibition of ALDH1A but the mechanism of irreversible inhibition of ALDH1A2 by WIN18,446 is not known. A crystal structure of ALDH1A2 in complex with WIN18,446 was solved in which a derivative of WIN18,446 was found covalently adducted to the active-site cysteine. Inspection of the structure suggested that the derivative is unstable and may be an intermediate stabilized under crystallographic conditions. In the current work we tested this hypothesis. We identified and characterized an aldehyde metabolite of WIN18,446, which we designated M-54. M-54 is likely the metabolite of the intermediate observed in the crystal structure. We then identified WIN18,446-ALDH1A2 protein using a range of proteomics techniques. Our data shows that WIN18,446 forms a 292.07 Da modification on cysteine 319 of ALDH1A2, an adduct mass potentially resulting from the metabolic intermediate. Using this mass, we probed human liver samples from multiple donors and found WIN18,446 specific adducts in ALDH1A1 and ALDH2 active site cysteines. The current study provides new insight into the metabolism of WIN18,446 and the mechanism of inhibition of ALDH1A2 and demonstrates a proteomics workflow for identifying and validating drug–protein adducts of unknown mass.