Aim: A designed, improved, and validated isocratic High Performance Liquid Chromatography method was used to study mahanimbine. Background: The method is evaluated for linearity, range, system applicability, LOD, LOQ, accuracy, and precision. Materials and Methods: For chromatographic separation, an Agilent ZORBAX Bonus RP C-18 (250x4.6 mm, 5 mu) column is employed. The mobile phase contains Methanol: 0.1 % Triethylamine (93:07%). The flow rate is 1.2 ml /min. The method is used to estimate Mahanimbine by using modern extraction techniques. Results: Mahanimbine has 4.705 minutes of retention time at 254 nm. The calibration curve was linear at concentrations between 4 and 24 ng/ml (R2-0.999). Accuracy and precision is found within the limit. The detection and quantification limit are 0.02044 and 0.06194 respectively. The robustness of the developed method is demonstrated. Mahanimbine considerably deteriorated when exposed to water, 0.1N HCl, 0.1N NaOH, oxidizing agent, and UV radiation. Conclusion: Murraya koenigii is extracted using a different technique, including Soxhlet assisted extraction, Ultrasound assisted extraction, and Accelerated solvent extraction, and mahanimbine was evaluated in those extracts. A new HPLC technique that is precise, effective, and accurate has been developed. To estimate the Mahanimbine in Murraya koenigii, the method is successful.