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Steroid hormones are crucial substances that mediate a wide range of vital physiological functions. Because of the important biological significance of steroids, this paper presents a new targeted metabolic method based on adding stable isotope tags to hydroxyl containing and carbonyl containing steroid hormones with two pairs of synthesized derivatization reagents: deuterium 4-(dimethylamino)-benzoic acid (D-4-DMBA), and D-5-Girard P (D-5-GP) using of ultra performance liquid chromatography-multiple reaction monitoring (UPLC-MRM). Firstly, an Oasis PRiME hydrophilic-lipophilic balance (HLB) 96-well solid phase extraction plate was used to pretreat a number of biological samples simultaneously. Secondly, hydroxyl and carbonyl steroids were labeled using two pairs of synthetic reagents, namely DMBA and D-4-DMBA, and GP and D-5-GP, respectively. Thirdly, the mixed products were detected using UPLC-MRM and the mass spectroscopy conditions were optimized. Methodology development showed that the sensitivity was enhanced 1 to >500-fold. Finally, the new method was applied to analysis of urine samples of healthy males, females and rats. The results revealed that the method can be sensitive and reliable for simultaneous quantification of steroid hormones containing hydroxyl and carbonyl groups in 12 min in a single run. This method provided a powerful tool for studying the metabolic mechanism of steroids and contributed to the development of targeted metabolomics.