Background: Four major degradation products (1-4) of pralatrexate injection were formed under hydrolytic and light stress conditions. The impurities 1 and 2 were the potential photo degradation products and the impurities 3 and 4 were the potential hydrolytic degradation products. Objective: To prepare and characterize the novel degradation impurities 1, 2, 3 and 4 of pralatrexate injection using NMR, HR MS and IR techniques; and to develop and validate stability indicating anal. reverse phase HPLC-UV method for quant. simultaneous determination of potential degradation impurities, related substances of pralatrexate and pralatrexate active in pralatrexate liquid formulation. Methods: Gradient HPLC-UV method was developed for the quantification of degradation impurities, related substances and pralatrexate in pralatrexate injection. The separation was achieved on C18 column (250 mm X 4.6 mm, 5μm) using a mobile phase composed of sodium dihydrogen phosphate monohydrate in water (pH 3.0; 0.01M) and methanol. The components were monitored by the UVvisible detector at 242 nm with a flow rate of 1.0 mL/min. Results: The method validation parameters such as accuracy, selectivity, linearity, LOD, LOQ, precision, ruggedness and robustness were demonstrated successfully for pralatrexate and its degradation impurities. The stability-indicating capability of the developed HPLC method was demonstrated by adequate separation of all potential pralatrexate related substances from pralatrexate stressed drug product samples. Conclusion: The developed stability indicating HPLC method was found to be suitable for the simultaneous quant. determination of potential degradation impurities and related substances of pralatrexate and pralatrexate active in pralatrexate liquid formulation.