In order to conveniently and rapidly isolate by group both conjugated and unconjugated serum androgens, a scheme has been devised for their differential extraction from commercially available, disposable octadecylsilane cartridges (Sep-Pak C18). Using added radioactive steroid standards and detection of endogenous serum steroids by group-specific enzymatic assays, the quantitative recovery of steroid glucuronides and sulfates in the 47% methanol fraction and of unconjugated steroids in the 100% methanol fraction was observed. Maximum recovery of serum protein-bound steroids (e.g. testosterone) was achieved with serum denatured by urea and heat. In order to separate glucuronides from sulfates, sequential hydrolysis of the conjugated fraction (47% methanol) by enzymatic hydrolysis and then organic solvolysis as well as an additional Sep-Pak cartridge extraction step was required. Groups of extracted steroids may be further separated and assayed by any appropriate method(s). An application is given which employs HPLC and an enzymatic assay for 17 beta-hydroxy- and 17-oxo-steroids to provide separate profiles of unconjugated, glucuronidated, and sulfated androgens in human, male serum.