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2-hydrazinyl-1,3-benzothiazole (HBT), 4-amino-4H-1,2,4-triazole (AT) and 2-hydrazinyl-1H-benzimidazole (HBI) were evaluated in this study as novel derivatization agents for the determination of steroid hormones in urine. The derivatization reactions were based on the interaction between the carbonyl group of steroids and the primary amino group of novel reagents. Optimal conditions for the derivatization of steroid hormones with novel agents, including pH, agent concentration, temperature and time of reaction were established. The resulting derivatives were separated and detected using reversed-phase ultra-high performance liquid chromatography–quadrupole time-of-flight mass spectrometry. The methods developed for achieved detection limits of 0.015–0.15 ng/mL for HBT derivatives, 0.15 ng/mL for AT derivatives and 0.075–0.15 ng/mL for HBI derivatives, respectively. During the reaction with these new agents, both syn- and anti-forms of derivatives of steroid hormones are formed, similar to other hydrazines. Based on the molecular properties of the obtained derivatives, the influence of substituents on the chromatographic characteristics was studied. The procedure was successfully applied to the analysis of authentic urine samples, and the results were compared with those obtained from hydroxylamine-based and Girard’s reagent T-based derivatization, demonstrating the prospects for further study of new reagents.