Chromatographic behavior of a new antifungal drug, (2-[(Z)-1-(3,5-diphenyl-1,3,4-thiadiazol-2(3H)-ylidene)-methyl]-3,5-diphenyl-1,3,4-thiadiazol-3-ium chloride (TDZ), and its impurities was studied using several types of columns, mobile phases and elution modes. A new reversed phase high-performance liquid chromatography (HPLC) method for the determination of TDZ and its impurities in a pharmaceutical substance and dosage form (tablets, 60 mg) was developed and validated. The optimal chromatographic separation of TDZ and its impurities was performed on a 5-μm C8 column (4.6 mm × 250 mm, I.D.). The mobile phase consisted of 0.03% TFA in water (pH 2.5) and acetonitrile under gradient elution condition (from 40:80 v/v to 80:20 v/v for 20 min) at a flow rate of 1.0 mL min−1. UV detection was set at a wavelength of 250 nm. The limit of detection (LOD) and quantification (LOQ) were 0.05 and 0.1 μg mL−1, respectively. The method showed good linearity (r2 > 0.999) over the assayed concentration range (0.1–1.4 μg mL−1 for quantification of impurities and 0.005–0.013 mg mL−1 for quantification TDZ) and demonstrated good intra- and inter-day precision and accuracy. The method was validated and successfully applied for quantitative determination of TDZ and its impurities in both the pharmaceutical substance and tablets.