A simple, sensitive, selective and reproducible method based on reversed-phase chromatography was developed for the determination of ivermectin in human plasma. The internal standard (moxidectin) was separated from ivermectin on a Hypersil Gold 018 column (150 × 4.6 mm, 5 μum particle size), with retention time of 3.7 and 7.0 minutes, respectively. Fluorescence detection was set at an excitation and emission wavelength of 365 and 475 nm, respectively. The mobile phase consisted of acetonitrile, methanol and distilled water (50:45:5, v/v/v), running through the column at a flow rate of 1.5 ml/minute. The Chromatographic analysis was operated at 25°C, Sample preparation (100 pi plasma) was done by a single step protein precipitation with acetonitrile, followed by derivatization with 100 μl of N-methylimidazole solution in acetonitrile (1:1, v/v) and 150 μl of trifluoroacetic anhydrous solution in acetonitrile (1:2, v/v). Calibration curve over the concentration range of 20-8,000 ng/ml plasma was linear with correlation coefficient better than 0.995. The precision of the method based on withinday repeatability and reproducibility (day-to-day variation) was below 15% (coefficient of variation) Good accuracy was observed for both intra-day and inter-day assays, as indicated by the minimal deviation of mean values found with measured samples from that of the theoretical values (below +15%). Limit of quantification was 0.02 ng using 100 μl sample. The mean recovery for ivermectin and the internal standard was greater than 90%. The method was free from interference from endogenous substances and commonly used drugs. The method appears to be robust and has been applied to the investigation of plasma concentration vs time profile of ivermectin in five healthy Thai volunteers following a single oral dose of 200 μg ivermectin/kg body weight.