Objective: High performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC/MS) methods were developed for the determination of ganciclovir and its related substances. Methods: A Hypersil ODS2 column (4.6 mm x 250 mm, 5 μm) was used with a mobile phase of 0.02 M potassium dihydrogen phosphate buffer (pH 6.0)-methanol (92:8) at a flow rate of 1.0 mL/min, and UV detector set at 254 nm was used for monitoring the eluents. Results: The method was simple, rapid, selective and capable of separating all related substances at trace level with a detection limit of 0.04 μg/mL. It has been validated with respect to accuracy, precision, linearity, and limits of detection and quantification. The linearity range was 10.2-153.0 μg/mL with r = 0.9998. The percentage recoveries ranged from 96.7% to 101.6%, and RSD was 1.24%-1.96% (n = 5). Conclusion: The method was found to be suitable not only for monitoring the reactions during the process development but also for quality control of ganciclovir. For identification of related substances, LC/MS was used. The mainly related substances of ganciclovir active pharmaceutical ingredients (API) were determined as guanine, (1, 3-dioxolan-4-yl) methyl acetate, and diacetyl guanine.