An analytical method based on on-line SPE-LC-APCI-MS/MS has been developed for the detection and quantification of eight selected estrogenic and progestagenic steroid hormones; estrone (E1), 17β-estradiol (E2), estriol (E3), 17α-ethinylestradiol (EE2), levonorgestrel (LEVO), medroxyprogesterone (MEDRO), norethindrone (NORE) and progesterone (PROG) in wastewater matrices. The injection volume could range from 1 to 10-mL according to the expected concentration of steroid hormones in matrix. The method characteristics are: analysis time per sample (<15 min), acceptable recovery values (71-95%), good precision (RSD ≤ 10%) and limits of detection at the low-nanogram per liter levels in affluent and effluent wastewaters (8-60 ng L(-1)). In particular, a detailed discussion of optimization parameters impacting overall performance of the method has been presented (sample collection, filtration and storage). All optimization and validation experiments for the on-line SPE method and chromatographic separation were performed in environmentally-relevant wastewater matrices. This method represents a compromise between analysis time, higher sample throughput capabilities, sample volume and simplicity for the analysis of both progestagenic and estrogenic steroid hormones in a single run, with LODs and LOQs sufficiently low to detect and quantify them in environmental wastewater matrices. Thus, the applicability of the method was tested on affluent and effluent wastewaters from two wastewater treatment facilities using different processes (biological and physico-chemical) to evaluate their removal efficiency for the detected steroid hormones.