Methotrexate is an antimetabolite and antifolate drug widely used in the treatment of cancer, rheumatoid arthritis, psoriasis, and other autoimmune disorders. The present study was aimed at developing and validating a simple, rapid, accurate, and precise Reverse Phase High Performance Liquid Chromatography (RP-HPLC) method for the estimation of Methotrexate in bulk drug form. Chromatographic separation was achieved using a C18 column with a suitable mobile phase consisting of buffer and organic solvent in optimized proportion at a specific flow rate. Detection was carried out using a UV detector at an appropriate wavelength. The developed method showed good peak symmetry with satisfactory retention time for Methotrexate. The method was validated according to International Council for Harmonisation guidelines for various validation parameters including linearity, accuracy, precision, specificity, robustness, limit of detection (LOD), and limit of quantification (LOQ). The calibration curve demonstrated good linearity within the selected concentration range with a high correlation coefficient value. Accuracy studies indicated satisfactory recovery, while precision studies showed low percentage relative standard deviation, confirming the reproducibility of the method. The proposed RP-HPLC method was found to be simple, economical, sensitive, and reliable for routine quantitative analysis of Methotrexate in bulk pharmaceutical formulation and quality control laboratories.