Vishamusthi (Strychnos nux-vomica Linn.), a medicinal plant described as Upavisha (semi-poisonous) group of Ayurvedic Pharmacopoeia of India. Vishamusthi has widely been used and being practiced in several illness namely nervous debility, paralysis, weakness of limbs, sexual weakness, dyspepsia and etc. Ayurveda practices strictly recommend the use of Vishamusthi in therapeutics only after proper shodhana (purificatory procedure) through specific medias such as Gomutra (cow’s urine), Godugdha (cow’s milk), Goghrita (cow’s ghee), and etc. Although various shodhana procedures are recommended in Ayurvedic treatise, but updated scientific researches regarding the shodhana methods are lacking. The present study was undertaken to investigate the physicochemical and phytochemical parameters, quantitative estimation of brucine using cutting edge research tools such as high-performance thin layer chromatography (HPTLC), liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) analyses of Vishamusthi seeds before and after purification. The remarkable changes have been observed in different physicochemical parameters, HPTLC, HPLC, GC-MS and LC-MS chromatographic profiling before and after shodhana process of Vishamusthi seeds. Quantitative HPLC studies revealed that the process of shodhana resulted in depletion of toxic brucine (chief poisonous constituent of Vishamusthi seeds) reduced to 79.66% in chloroform extract and 64.54% in ethanol extract after shodhana process.