A derivatization procedure and a high-performance liquid chromatographic (HPLC) method of analysis for pilocarpine are described. The method is based on the quaternization of the 3'-tertiary amino group of the methylimidazole ring of pilocarpine with p-nitrobenzyl bromide. The HPLC system employs and RP-ODS column with a methanol-water mobile phase containing octanesulfonate as an ion-pairing agent. The sensitivity of the method permits the analysis of pilocarpine in biological fluids such as aqueous humor. The method is selective for pilocarpine in the presence of isopilocarpine. Its applicability to the analysis of aromatic heterocyclic and alkyl tertiary amines is demonstrated.