BACKGROUND Many hormones show the effects of protein assimilation and growth promotion, and they are frequently used as veterinary drugs in livestock, which has harmful effects on human health. It is necessary to determine their contamination level in animal-derived food, especially in milk. OBJECTIVE In this study, a detailed procedure is described for an automated online solid-phase extraction (SPE)-HPLC method capable of detecting five hormones (i.e., estriol, prednisone acetate, hydrocortisone, diethylstilbestrol, and estrone) in cow milk. METHODS The corresponding milk samples were precipitated by addition of acetonitrile and then purified as well as enriched by a polar-enhanced polymer (PEP) online SPE column. The supernatants were directly injected into the online SPE-HPLC system using methanol-water as the mobile phase mixture. RESULTS The linearity range of the method was 0.1-25 μg/mL for prednisone acetate, hydrocortisone, and diethylstilbestrol, 0.2-25 μg/mL for estriol, and 0.5-25 μg/mL for estrone, with correlation coefficients (r) ranging from 0.9994 to 0.9996. The recovery rates determined at three concentration levels for the five compounds were in the range of 70.82-112.90%. LODs of estriol, prednisone acetate, hydrocortisone, diethylstilbestrol, and estrone were 0.023, 0.005, 0.006, 0.004, and 0.054 μg/mL, respectively. CONCLUSIONS This automated online SPE-HPLC method was both effective and reliable in the simultaneous measurement of five hormones, and the method was successfully applied to the detection of five hormone species in milk. CONCLUSIONS An automated online SPE-HPLC method has been developed for the analysis of five hormones in cow milk. Online SPE proved to be a powerful technique for determining five hormones simultaneously. This method ensured simple sample pretreatment and less operation time. The established method was successfully applied to the analysis of five hormone species in milk.