<p>Steroid hormones play crucial regulatory roles in human growth, development, reproduction, and the maintenance of internal environmental homeostasis. Because the concentrations of most hormones in the body are extremely low and their chemical properties vary widely, developing an accurate and sensitive quantitative method has become a primary objective in clinical diagnostics. In this study, an isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method was developed and validated for the simultaneous quantification of 22 steroid hormones in human serum. To enhance extraction and purification efficiency, a protein precipitation–solid-phase extraction (PP-SPE) approach was employed, forming a “dual purification” strategy. The limits of quantification for progesterone, cortisol, dihydrotestosterone, and dehydroepiandrosterone sulfate were 0.05, 0.50, 0.05, and 1.00 ng/mL, respectively, representing an improvement over previously reported values obtained using similar sample pretreatment methods. Method validation results demonstrated strong linearity for all 22 steroid hormones (<i>R</i>² &gt; 0.98). Accuracy ranged from 86.9% to 119.5%, intra-day precision was below 9.7%, inter-day precision was below 12.0%, and the matrix effect was controlled within 80.3%–118.1%. These findings indicate that the method is accurate, reliable, and suitable for analyzing multiple components in complex serum matrices. Twenty real serum samples were analyzed to evaluate the clinical applicability of the method. The results demonstrated that this method can be used clinically to monitor steroid hormone levels, assist in diagnosing and treating endocrine disorders, and provide a diagnostic basis for adrenal diseases. It holds significant reference value for clinical laboratory testing.</p> Graphical Abstract <p></p>