A simple, linear, rapid, precise and stability-indicating analytical method was developed for the estimation of related substances and degradants of Doxorubicin hydrochloride API and tablets. The chromatographic separations were achieved using high performance liquid chromatograph (X-Terra C18, 100mm X 4.6mm, ID with 3.5 microns particles column) employing mixture of 0.05mM ammonium acetate, methanol and Acetonitrile (500:250 :250v/v) as mobile phase in an isocratic elution mode with a 0.5 mL/min flow rate was chosen. Two impurities were eluted within ten minutes of run time. The column temperature was maintained at 25ºC and a detector wavelength of 254 nm was employed. Doxorubicin HCl was exposed to acid, base thermal, photolytic, and oxidative stress conditions. The stressed samples were analyzed by the proposed method. The degradation of Doxorubicin hydrochloride was observed under Acid hydrolysis, base hydrolysis and oxidative hydrolysis. The drug was found to be stable in all other stress conditions applied. Successful separation of the drug from organic impurities and degradation products formed under forced degradation was achieved. The developed HPLC method to determine the related substances and assay of Doxorubicin HCl can be used to evaluate the quality of regular production samples. It can be also used to test the stability samples of Doxorubicin HCl.