Abstract The electrochemical behavior of the over-the-counter antihistamine drug pyrilamine and its N-oxide analogue, have been studied by several voltammetric methods. Cyclic voltammograms of pyrilamine maleate in 0.1 M ammonium acetate at pH 7.0 indicated a quasi-reversible electrode process by observing a wave at +0.85 V and +1.30 V in the initial anodic sweep followed by a wave at -1.30 V versus Ag/AgCl. Differential pulse and hydrodynamic voltammetry of pyrilamine and the N-oxide were examined to determine oxidation potentials for use in high-performance liquid chromatography with electrochemical detection (HPLC-ED). Differentiation between pyrilamine and its N-oxide was achieved in HPLC-ED analyses at a detection potential of +0.7 V and +0.9 V versus Ag/AgCl with tandem ultraviolet detection at 254 nm. Utility of the HPLC-ED method was demonstrated by the analysis of pyrilamine and the N-oxide in microbial biotransformation samples.