The plant, Orthosiphon stamineus (cat whiskers) is a medicinal herb belonging to the family Lamiaceae, and the plant leaves are commonly used as the herbal tea, popularly known as Java tea. It is used as a diuretic agent and for treatment against heat rheumatism. Currently, there are no protein profile data available for this important plant species. This study focused on the optimization of total protein extraction using four different extraction methods; QB buffer, phenol/sodium dodecyl sulfate with three pre-washed steps, phenol/sodium dodecyl sulfate without pre-wash steps and sigma protein extraction kit, it is aimed at determining the best protein extraction method, determine patterns and theoretically identify proteins. Overall, phenol/sodium dodecyl sulfate with three pre-wash steps result in better protein quality and pattern of separation. A total of 104 functional proteins were identified, each containing at least one unique peptide. Among the distinct proteins, rubisco activase and triosephosphate isomerase correspond to the chloroplastic protein of photosynthesis/carbohydrate metabolism, while phosphoglycerate kinase and glyceraldehyde are cytosolic enzymes of glycolysis pathway which were found to be significant housekeeping proteins in of the leaf tissue. The result of this study will be useful for advanced pharmaceutical research and could serve as a baseline for further proteomics work on O. stamineus and similar plant species.