Metabolic stability, a crucial aspect in drug development, reflects a compound’s resistance to metabolic degradation. Early-stage evaluation of a drug candidate’s metabolism can substantially lower the risk of failure in the development pipeline. These studies are predominantly conducted in vitro using microsomal enzymes, which play a significant role in drug metabolism. Among the various analytical techniques available, liquid chromatography is most commonly employed. In this study, we assessed the metabolic stability of a previously synthesized hydrazone-sulfonate derivative known for its biological activity. The compound’s metabolic stability was investigated using LC-MS/MS in vitro on rat liver microsomes. Analyses were conducted at different incubation time points: 0, 5, 10, 15, 30, and 60 minutes. Results demonstrated that the compound’s stability was highly dependent on the presence of cofactors. It remained stable in the absence of cofactors and under buffer-only conditions.