Steroids are essential hormones that play a crucial role in homeostasis of many biological processes including sexual development, spermatogenesis, sperm physiology and fertility. Although steroids have been largely studied in many biological matrices (such as urine and plasma), there is very limited information of the steroid content and their study as potential indicators of the quality of the seminal fluid. In this study, a LC-HRMS (liquid chromatography-high resolution mass spectrometry) strategy has been developed in order to obtain the extended steroid profile of human seminal fluid. A comparison between supported liquid extraction (SLE) and solid liquid extraction (SPE) was carried out and the chosen SPE method was further optimized to evidence the largest possible number of compounds. Steroids were automatically annotated by using DynaStI, a publicly available retention time prediction tool developed in our lab, to match the experimental data (i.e. accurate mass and tR). Altogether, these resources allowed us to develop a post-targeted approach able to consistently detect 41 steroids in seminal fluid (with half of them being androgens). Such steroid pattern was found to be stable across different extraction times and injection days. In addition to accurate mass and retention time, the identity of 70% of the steroids contained in such steroid profile was confirmed by comparing their fragmentation patterns in real samples to those of pure commercial standards. Finally, the workflow was applied to compare and distinguish the steroid profile in seminal fluid from healthy volunteers (n = 7, with one of them being a vasectomized subject). In all, the developed steroidomics strategy allows to reliably monitor an extended panel of 41 steroids in human seminal fluid.