A high-performance liquid chromatographic assay is described for the separation and quantification of nadolol isomers in human plasma. The isomers were quantified using reverse-phase HPLC and fluorometric detection after derivatization with the chiral reagent R(-)-1-(naphthyl)ethylisocyanate [R(-)-NEI]. The N-isopropyl analogue (one isomer) of nadolol was used as the internal standard. The method was reproducible based on precision studies where the percent relatives standard deviation was less than 15%. The lower limit of quantitation for each isomer was 2.5 ng/mL. This method was used to evaluate the pharmacokinetic profile of nadolol isomers in human subjects following both single and multiple oral dosing.