A high-performance liquid chromatographic method and a UV spectrophotometric method for the quantitative determination of Pemetrexed disodium, a highly potent antifolate, in powder for infusion were developed in present work. The parameters linearity, precision, accuracy, robustness, limit of detection and limit of quantitation were studied according to International Conference on Harmonization guidelines. The HPLC was carried out by reversed-phase technique on Kromasil C18 (250 × 4.6 mm, 5 ?m) column with a mobile phase composed of 20 mM dibasic phosphate buffer (adjusted to pH 6.50 with ortho-phosphoric acid) and acetonitrile (88:12; v/v). The samples were prepared in water and the stability of Pemetrexed disodium in aqueous solution at 25°C was studied. The results were satisfactory with good stability after 24 hr at 25°C. UV spectroscopic determination was carried out at an absorption maximum of 225 nm using distilled water as solvent. Statistical analysis by Student’s t - test showed no significant difference between the results obtained by the two methods. The proposed methods are simple, rapid, precise and accurate and can be used for the reliable quantitation of Pemetrexed disodium in pharmaceutical formulation.