A simple and specific method for determination of methyltestosterone (MT) has been established by a gas chromatography/mass selective detector and applied in plasma of healthy male volunteers received a single oral dose of 50 mg MT $(Testo^{TM};tablets,;25;mg)$ for bioavailability test. This method involves using liquid-liquid extraction of the sample with diethyl ether and derivatization with MSTFA. MT showed good resolution in this condition. The detection limit of quantitation was 5 ng/ml. A good linearity (r>0.996) was obtained at the range of 5-250 ng/ml of MT. Intra-day precision and accuracy were 2.76-12.56% and 0.39-8.01 %, and inter-day precision and accuracy were 2.29-17.69% and 0.42-7.99%, respectively. The established method was applied on bioavailability test of MT in human volunteers. The value of $AUC_{0;to;last}$ to last was $264.5{pm}123.9;ng{cdot}hr/ml$ and that of $AUC_{0;to;inf}$ was determined to be $275.2{pm}126.5;ng{cdot}hr/ml$. The values of $C_{max}$ and $T_{max}$ were $95.9{pm}67.1;ng/ml$ and $1.13{pm}0.9;hr$, respectively. The mean elimination half-life $(t_{1/2})$ was $4.4{pm}0.9;hr$. This analytical method is suitable and useful for the pharmacokinetics and bioequivalence studies of MT.