The aim of the current study is to investigate thethin layer chromatography (TLC) and gas chromatography and mass spectroscopy analysis (GC-MS) of methanolic extract of Acacia ferruginea thorns. The bioactive molecules were determined by qualitative TLC and GC-MS method. In TLC exhibited maximum 0.6 retention factor (RF) value of the plant extract in F254 wavelength in dark blue colour, F366 wave length and in visible light not shown any peaks and RF values. In the GC-MS analysis, 37 bioactive molecules were exhibited and in that 10 are in higher concentration by the retention time and their % of peak and area covered in the analysis compared to other chromatograms of the fractions. Important compounds identified as Methyl mannose (57.14), Phenol, 2-methoxy-3-(2-propenyl)- (4.85), stigmasterol (4.65), 1-Hexacosanol (3.83), Lupeol (3.60), gamma Sitosterol (3.52), Phenol, 4-[2-(dimethylamino)ethyl]-(3.17), Ergost-5-en- 3-ol, (3.beta.)- (2.53), Oleic acid (2.44), Oleoyl chloride (1.53), Sucrose (1.82). The presence of these bioactive molecules in the plant extract may provide the scientific evidences for the cytotoxic effect, insecticidal and other biological properties.