Orchids are highly valued for their therapeutic properties. Malaxis acuminata (vernacular name: Jeevak), is a vanishing orchid that has mystical healing potential. Wavelength dispersive X-ray fluorescence (WD-XRF) spectroscopy was employed to conduct elemental analysis of this orchid plant. Elements Potassium (K), Calcium (Ca), Phosphorus (P), Magnesium (Mg), Silicon (Si), Sulphur (S), Chlorine (Cl), Aluminium (Al), Iron (Fe), Ruthenium (Ru), Manganese (Mn), Zinc (Zn), Rubidium (Rb), Titanium (Ti), Copper (Cu), Strontium (Sr), and Nickel (Ni) are documented quantitatively and qualitatively. These elements are pivotal in shaping the plant's medicinal potential. Likewise, a multitude of organic molecules within the plant exhibit therapeutic potential. Stigmasterol is one such molecule, esteemed for its remarkable properties and a spectrum of health benefits. The present investigation additionally encompassed the establishment and validation of a methodology for the precise quantification of stigmasterol within the petroleum ether extract, utilizing high-performance thin-layer chromatography (HPTLC). The bioactive compound was isolated through the utilization of a specific mobile phase of toluene: ethyl acetate: methanol (8:1.5:0.5, v/v/v), and an optimal wavelength of 580 nanometers was ascertained. Validation of this method was conducted by the guidelines established by the International Council for Harmonisation (ICH). The calibration range for the stigmasterol was established to span from 400 to 3600 micrograms per spot (μg/spot). The Rf (retention factor) value for the separation of stigmasterol was determined to be 0.56 ± 0.04. The limit of detection (LOD) and limit of quantification (LOQ) were established at 15.954 nanograms per spot (ng/spot) and 48.344 ng/spot, respectively. Linearity (r2 0.999), accuracy (98.14– 99.21 %), and RSD of precision (1.953–2.984). specificity and robustness were evaluated by ICH guidelines. The stigmasterol content was 634.6 ± 3.28 μg/ml, which demonstrates promising results. This method exhibits substantial applicability in pharmaceuticals, nutraceuticals, food, and health-related sectors.