A rapid, sensitive and selective analytical method was developed and validated for the determination of compound K, a major intestinal bacterial metabolite of ginsenosides in human plasma. Liquid-liquid extraction was used for sample preparation and analysis, followed by liquid chromatography tandem spectrometric analysis and an electrospray-ionization interface. Compound K was analyzed on a Phenomenex Luna C18 column ($100{ imes}2.00$ mm, 3 ${mu}m$) with the mobile phase run isocratically with 10 mM ammonium acetate-methanol-acetonitrile (5:47.5:47.5, v/v/v) at a flow rate of 0.5 mL/min. The method was validated for accuracy (relative error <12.63%), precision (coefficient of variation <9.14%), linearity, and recovery. The assay was linear over the entire range of calibration standards i.e., a concentration range of 1 ng/mL to 1,000 ng/mL ($r^2$ >0.9968). The recoveries of compound K after liquid-liquid extraction at 1, 2, 400, and 800 ng/mL were $106.00{pm}0.08%$, $103.50{pm}0.19%$, $111.45{pm}5.21%$, and $89.62{pm}34.46%$ for intra-day and $85.40{pm}0.08%$, $94.50{pm}0.09%$, $112.50{pm}5.21%$, and $95.87{pm}34.46%$ for inter-day, respectively. The lower limit of quantification of the analytical method of compound K was 1 ng/mL in human plasma. The developed method was successfully applied to a pharmacokinetic study of compound K after oral administration in ten of healthy human subjects.