:  There is an increasing demand for natural-based remedies due to their safety and efficiency compared to synthetic-based medicine. In addition, the unique bioactive properties of Mitragyna speciosa has also attracted interest in their potential application for pharmacological purposes. Therefore, this study aimed to optimise the Ultrasound-Assisted Extraction (UAE) method using Response Surface Methodology (RSM) for the production of crude methanolic extract from Mitragyna speciosa leaves. Statistical optimisation was performed to evaluate the effect of temperature, extraction time, and volume of solvent. The phenolic and flavonoid contents of crude methanolic extract were analysed through Total Phenolic Content (TPC) and Total Flavonoid Content (TPC), respectively while Gas Chromatography-Mass Spectrometry (GC-MS) was carried out to determine the characteristic of the crude methanolic extract. The optimal conditions at a temperature of 34 °C, extraction time of 25 min, and volume of the solvent of 166 mL resulted in a maximum yield of 49.72% of the crude methanolic extract with TPC and TFC recorded at 137.3 ± 15.7 mg GAE/g and 90.3 ± 15.3 mg RE/g, respectively. The GC-MS analysis identified the presence of 18 phytocompounds in the crude extract including glycerine, phytol, and stigmasterol. These identified compounds could exhibit certain therapeutic effects such as anti-inflammatory, antioxidant, and antibacterial effects, therefore strengthening the suggestion that the various bioactive compounds contributed to the bioactive properties in M. speciosa besides alkaloid, subsequently applied as an effective traditional herbal medicine to treat various illnesses. 

Keywords: Response surface methodology; Mitragyna speciosa; Total phenolic content; Total flavonoid content; phytocompounds; GC-MS