The separation of dacarbazine and its two related impurities, 5-amino-imidazole-4 carboxamide (AIC) and 2-azahypoxanthine (AHX) was investigated by capillary electrophoresis and high performance liquid chromatography. Baseline separation of dacarbazine and its related impurities was achieved by capillary electrophoresis (CE) on a fused silica capillary (70 cm × 50 μm, i.d) with an electrolyte buffer of methanol: phosphate buffer (0.025 M, pH 4) - 2:98 v/v at an applied voltage of 15 kV and detection at 220 nm. The CE method could not be used for the quantitation of impurities because at high concentration, the dacarbazine peak overlapped the nearest impurity peak. The initial HPLC method developed on an octadecylsilane (ODS) column successfully separated the impurities from the dacarbazine, but required a longer time of analysis (>50 min). Another HPLC method developed on an avidin protein column separated the drug and its impurities with a run time of less than 27 min and was chosen for method development and validation.