A highly sensitive analytical tool for the fast quantification of irsogladine in human plasma was developed. Cleanup using a solid-phase extraction technique is a simple method for extracting both irsogladine and lamotrigine (internal standard) spiked into human plasma. The resolvable separation of both analytes through reversed-phase high-performance liquid chromatography (HPLC) was carried out within 5 min. The HPLC–electrospray ionization (ESI)–tandem mass spectrometry (MS/MS) method, which was operated in a selected reaction monitoring mode specific to the target analytes, was verified for use in the quantification of irsogladine. The inter- and intra-day precision (relative standard deviation, RSD) of irsogladine spiked into quality control samples were <7%, and their accuracies were between 96.6% and 102.1%. The calibration curve for irsogladine spiked into human plasma was linear over the range from 1.8 to 100 ng mL −1 with lower limit of quantification at 1.8 ng mL −1. The established method was successfully applied for a bioequivalence study of irsogladine.