The extraction and untargeted UHPLC-HRMS analysis of endogenous steroids in serum samples is described in this protocol. The employed full-scan acquisition mode provides the adequate sensitivity to highlight the main endogenous steroids present in blood, including mineralocorticoids, progestogens , and androgens. Technical aspects for both chromatography and mass spectrometry are discussed in detail, together with a proposition of setup for sample sequence and data analysis. Furthermore, general comments are given to help the assessment of data quality and system performance.