An HILIC method was developed to quantitatively determine trace amounts of 1,2,4-triazole and 4-amino-1,2,4-triazole in fluconazole drug substance. Separation was achieved on HILIC column using isocratic elution with mixture of water, acetonitrile and phosphoric acid mobile phase and UV detection at 205 nm. The method was validated according to ICH guideline requirements and was determined to be linear in the range of 0.6–22.0 μg/mL. Method accuracy and precision were assessed by spiking 1,2,4-triazole and 4-amino-1,2,4-triazole at three levels over the range of 2.5–15 μg/mL, the recoveries ranged from 78.6% to 95.7% for 1,2,4-triazole and 94.6% to 103.4% for 4-amino-1,2,4-triazole. The intermediate precision was represented by the overall %RSD of 6.9% (n = 9) for 1,2,4-triazole and 3.6% (n = 9) for 4-amino-1,2,4-triazole. The method was also shown to be repeatable at concentration levels of 2.5, 12.5 and 15 μg/mL with n = 3 %RSD values of 3.7%, 1.0% and 1.3% for 1,2,4-triazole, and 0.3%, 1.2% and 0.6% for 4-amino-1,2,4-triazole. The method was found to be specific, stability-indicating and has a detection limit of 0.8 μg/mL and a quantitation limit of 2.5 μg/mL. Finally, the method was demonstrated to be robust to small variations of key chromatographic parameters.