A validated specific stability indicating reversed-phase HPLC method was developed for the quantitative determination of Amsacrine as well as its related substances determination in bulk samples, in presence of degradation products and its process related impurities. Forced degradation studies were performed on bulk sample of Amsacrine as per ICH prescribed stress conditions using acid, base, oxidative, thermal stress and photolytic degradation to show the stability indicating power of the method. Significant degradation was observed during basic hydrolysis, slight degradation was observed in oxidative and thermal stress and no degradation was observed in other stress conditions. The chromatographic method was optimized using the samples generated fromforced degradation studies and the impurity spiked solution. Good resolution between the peaks corresponds to process related impurities and degradation products from the analyte were achieved on Inertsil ODS column using the mobile phase consists a mixture of 1.0%Triethyl amine in 20mMPotassiumdihydrogenOrthophosphate pH adjusted to 6.5 with ortho phosphoric acid in water and Acetonitrile using a simple linear gradient. The detection was carried out at wavelength 248 nm. The mass balance in each case was in between 99.4% to 99.9% indicating that the developed LC method was stability-indicating. Validation of the developed LCmethod was carried out as per ICH requirements. The developed method was found to be suitable to check the quality of bulk samples ofAmsacrine at the time of batch release and also during its stability studies.