Icaritin (ICT), a bioactive metabolite of prenylflavonoids from genus Epimedium, has displayed potential benefits for the treatment of osteoporosis, prostate cancer, liver cancer, renal cancer and breast cancer. To investigate the quantity of ICT in bones in vivo, a simple and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed. After a rapid one-step liquid-liquid extraction using ethyl acetate with recovery more than 87.2% at four levels (0.1, 0.2, 8 and 15 ng/mL), ICT and internal standard coumestrol were analyzed on a C18 column using a gradient elution of acetonitrile and water containing ammonium formate and formic acid at a flow rate of 0.3 mL/min. Quantification was performed using selected reaction monitoring mode to monitor precursor-product ion transitions of m/z 367.1→297.1 for ICT and of 267.0→211.1 for coumestrol in the negative ionization mode. A calibration curve with good linearity (r>0.99) within the concentration range of 0.1-20 ng/mL for ICT was obtained with the lower limit of quantification of 0.1 ng/mL. Matrix effect did not interfere with ICT analysis and ICT was stable under three freeze-thaw cycles, short-term temperature, post-preparative and long-term temperature conditions. The method was successfully applied to a dynamic distribution of ICT in mouse bone after a single intraperitoneal administration to ICT to mice.