The commercially available fluorescence polarization immunoasay (FPIA) method for phenobarbital is demonstrated to be useful in the quantification of pentobarbital in serum. The effective range for the method is 1.0 to 80.0 mg/L. The method is simple, precise, and fast. The application of the method is limited to patients receiving pentobarbital as the only barbiturate. The FPIA is recommended for use in clinical laboratories when a rapid estimation of pentobarbital is required in monitoring patients undergoing high-dose pentobarbital therapy.