A method for determining concentration levels of ganaxolone in rat, monkey, dog and human plasma was validated in the range of 5-1500 ng/ml using a 200-microl plasma sample volume. This validation report describes the linearity, specificity. sensitivity, reproducibility, accuracy, recovery and stability of the analytical method. The inter-day C.V. ranged from 0.5 to 9.2%, intra-day C.V. from 0.7 to 8.8% and intra-day accuracy (mean absolute percentage difference) ranged from 0.0 to 14.0% for rat, monkey, dog and human plasma. The method was used for the routine analysis of ganaxolone in rat, monkey, dog and human plasma and summary of the pharmacokinetic data are presented.