In the present study, we effectively detected 10 steroids and glucuronic acid-conjugated steroid metabolites in 12 min by ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). Steroids testosterone (T), 5α-dihydrotestosterone (DHT), androsterone (ADT), etiocholanolone (ETIO), estradiol (E2) and their glucuronide conjugates were well-separated on an Eclipse Plus C18 column (2.1 mm×50 mm, RRHD 1.8 µm). The mobile phase consisted of a mixture of methanol and ultrapure water (containing 1 mM ammonium formate) at a ratio of 60:40 (v/v), and the flow rate was set at 0.25 mL/min. The LC eluate was detected by electrospray ionization (ESI) source in both positive and negative ion modes. Neutral loss (NL of 176, 194, 211 and 229 Da in positive mode) and precursor ion (PI of m/z 141, 159 and 177 in positive mode and 75, 85 and 133 in negative mode) methods were applied for the detection of steroid glucuronides. The multiple reaction monitoring (MRM) transitions were m/z 289.3→97.1, 291.3→105, 291.3→199.2, 273.2→145.4 and 255.2→159.1 for T, DHT, ADT, ETIO and E2 in positive mode, respectively; as well as m/z 463.3→85 for T glucuronide (T-G), m/z 465.3→75 for DHT glucuronide (DHT-G), ADT glucuronide (ADT-G), ETIO glucuronide (ETIO-G) and m/z 447.3→271 for E2 glucuronide (E2-G) in negative mode. In addition, the analytical method was also applied for the detection of steroid glucuronides in pooled human liver microsomes (HLM), which might serve as a basis for further investigation of steroid metabolism in vivo and in vitro.