[en] Highlights: • Mass spectrometry fragmentation behaviors of steroid hormones were studied and interpreted. • Diagnostic product ions were summarized for characterization of steroid hormones. • A total of 80 and 107 steroidal hormones were characterized or tentatively identified in male and female, respectively. -- Abstract: Steroid hormones, structural derivatives of cyclopentanoperhydrophenanthrene, play important roles in modulation of many physiological processes. Comprehensive characterization of steroid hormones is valuable for understanding the process of human life activities and even disease diagnosis. Hitherto systematical characterization of steroid hormones has been rarely investigated. Here, we presented an integrated method for human urine analysis based on ultra-high performance liquid chromatography-high resolution mass spectrometry in data-dependent acquisition mode with the following parallel reaction monitoring mode. To process the data acquired by two scan modes, a comparative study of standards’ fragmentation behaviors and diagnostic product ions (DPIs) were firstly conducted to facilitate the characterization of steroid hormones. The fragmentation behaviors, DPIs, elemental composition and double-bond equivalent were then simultaneously utilized for systematical characterization of steroid hormones in human urine. Consequently, fragmentation pathways and DPIs for all types of steroid hormones were comprehensively interpreted. It is interesting to find that dehydration is not restricted in the form of hydroxyl groups loss, elimination of the carbonyl oxygen could also generate dehydrated ions. Ultimately, a total of 80 and 107 steroidal hormones were characterized or tentatively identified in human urine of male and female, respectively. The proposed method is expected to provide valuable insights for chemical characterization in complex matrixes.