<p>A sensitive and robust SPE–HPLC–MS/MS method was developed and validated for the simultaneous quantification of five steroid hormones in H295R cell culture medium. Chromatographic separation of the target steroids and selected potential endocrine‑disrupting chemicals (EDCs) was achieved using a ZORBAX Eclipse Plus C18 column (1.8 µm, 95 Å, 50 × 2.1 mm). Sample preparation was performed by solid‑phase extraction employing hydrophilic–lipophilic balanced polymeric reversed‑phase sorbent cartridges. Extraction recovery was evaluated at three concentration levels, tailored to each steroid, and exceeded 80% for all analytes. Intra‑ and inter‑day precision, expressed as relative standard deviation (RSD), ranged from 1 to 12%. Method limits of quantification were in the range of 8 and 73 pg mL⁻¹, enabling reliable detection of steroids present at low endogenous levels. In addition to method validation, the ESI response of steroids was systematically evaluated in the presence of the selected compounds. Under the reference chromatographic gradient, signal variations were generally limited, indicating stable and robust ionization performance during LC–ESI–MS/MS analysis. The validated method was successfully applied to the high-throughput analysis of 285 H295R cell culture medium samples, generating a dataset of 1425 individual hormone measurements obtained within a broader framework investigating steroidogenic responses to individual potential EDCs. The results confirmed the capability of the analytical method to reliably monitor hormone fluctuations across a broad concentration range, demonstrating its suitability for routine, large-scale sample analysis in complex in vitro systems.</p> Graphical abstract <p></p>