The genus Tribulus (Zygophyllaceae) comprises about 25 species commonly grows as herbs or shrubs in subtropical areas around the world. Tribulus longipetalus is an annual or biennial prostate herb, found in dry sandy soils in warm temperate zones of Egypt, Afghanistan, India and Pakistan. The fruit of this plant is used for the treatment of urinary disorders and cough. Literature survey revealed the presence of steroidal saponins, ligninamides, alkaloids and flavonoids from the genus Tribulus. Steroidal saponins with spirostan, furostane and cholestane nuclei and flavonol glycosides have also been reported from T. longipetalus. In our present study, we have isolated Longipetalosides A-C (1-3), three new furastan steroidal saponins together with a (25S)-5a-furastan-3β,22,26-triol (4) and a spirostanol steroid gitogenin (5) from methanolic extract of the whole plant of Tribulus longipetalis. These compounds were isolated through successive column chromatographic techniques after defating the water extract with petrol to get crude saponins fraction. The crude saponins fraction was further fractionated using silica gel column chromatography and CHCl₃-MeOH (1:0 to 1:1) as eluent to afford sub-fractions (1-6). Fraction 4 was subjected to repeated column chromatography, eluting with CHCl₃-MeOH-H₂O (5:1:0.1) to afford saponin fraction. This was further purified using Sephadex LH-20 column and MeOH as mobile phase. The final purification of saponins was carried out by HPLC using RP-18 column and MeOH and H₂O as mobile phase in different proportions to get pure saponins (1-3).The structures of these compounds (1-5) were established by using 1D (¹H, ¹³C) and 2D NMR (HMQC, HMBC, COSY, NOESY) spectroscopy, and mass spectrometry (ESIMS, HRESIMS), and in comparison with literature data reported for related compounds. Compounds 1-5 were evaluated for their inhibitory activities against enzymes a-glucosidase, lipoxygenase (LOX), acetylcholinesterase (AChE) and butyrylcholinesterase (BChE).