Temozolomide (TMZ) is a broad spectrum alkylating agent found effective in the treatment of glioblastoma multiforme, refractory anaplastic astrocytoma, and metastatic melanoma. The major drawback associated with TMZ is pH-dependent stability and short half-life. At physiological pH, it undergoes conversion to MTIC (methyltriazine imidazole carboxamide) and AIC (amino imidazole carboxamide), resulting in only 20–30% brain bioavailability. There is a need for an analytical method for the estimation of TMZ in stability samples and nanoformulations. In this research study, analytical methods were developed for the estimation of TMZ using two media pH 1.2 (0.1 N HCl) and pH 4.5 acetate buffer, which were validated for linearity, range, precision, accuracy, limit of detection, limit of quantification, and specificity as per ICH guidelines. The % RSD was found to be <2% indicating the reliability of the method. Further, the application of the developed methods was explored. The stability of TMZ in three pH conditions (1.2, 4.5, and 7.4) and the respective degradation rate kinetics was studied. Conversion of TMZ was found to follow first order kinetics with the conversion rate of 0.0011, 0.0011, and 0.0453 h⁻¹ in pH 1.2, 4.5, and 7.4 respectively. The developed methods accurately estimated the TMZ concentration in lipid nanoformulation (liposomes) indicated by ~100% recovery. Acetate buffer (pH 4.5) was found to be an appropriate dissolution media for TMZ loaded lipid nanoformulations. The developed methods were found to be suitable for routine analysis, for the determination of drug stability and estimation of temozolomide in lipid nanoformulations.