Icaritin is one of the Epimedium products with various biological activities. In the present study, we developed a rapid, reliable and robust UHPLC–MS/MS method to simultaneously determine unconjugated icaritin and its multiple glucuronides (icaritin-3-glucuronide, icaritin-7-glucuronide and icaritin-3,7-diglucuronide) in microsomal incubation systems, and applied it to study icaritin regioselective glucuronidation in vitro. We identified the involvement of human UDP-glucuronosyltransferase (UGT) isoforms in icaritin metabolism and further studied the kinetic profiles of icaritin glucuronidation using pooled human liver microsomes (HLMs), pooled rat liver microsomes (RLMs), pooled human intestine microsomes (HIMs) and UGTs, respectively. We also evaluated regioselective glucuronidation of icaritin by UGT isoforms and conducted time-dependent experiment to elucidate the metabolic pathways for icaritin clearance. Catalytic efficiency of microsomes is determined according to rank orders of total intrinsic clearance (CLint): CLint,HLM (24.19mL/mg/min)>CLint,RLM (13.15mL/mg/min)>CLint,HIM (6.43mL/mg/min). Besides, icaritin glucuronidation is mediated by multiple enzymes, with UGT1A1 the principal metabolizing enzyme (total CLint,UGT1A1=6.38mL/mg/min). As for the regioselectivity, except for UGT1A8 and UGT2B7, most UGT isoforms exhibit preference for the position of 3-OH on icaritin structure. Moreover, time-dependent conversion from monoglucuronides to diglucuronide indicate that icaritin-3,7-diglucuronide may be the final metabolite from icaritin elimination.