The stems of Cissus quadrangularis were collected systematically from the Coimbatore region of Tamil Nadu, India. Continuous ethanolic extraction using a Soxhlet apparatus yielded a crude ethanolic extract, which was subjected to percentage yield calculation for the extraction efficiency assessment. Phytochemical evaluation was performed using conventional thin-layer chromatography (TLC). Liquid Chromatography-Mass Spectrometry (LC-MS) and Gas Chromatography-Mass Spectrometry (GC-MS) analyses provided insights into the molecular constituents, whereas High-Performance Liquid Chromatography (HPLC) fingerprint analysis contributed to comprehensive characterization. The ethanolic extraction of C. quadrangularis using the Soxhlet technique resulted in a wet weight of the crude extract of 10 g, with a corresponding dry weight of 4.5 grams. The percentage yields calculated indicated that 2% of the wet ethanolic extract and 0.9% of the dry ethanolic extract were obtained from the crude drug. Phytochemical analysis revealed the presence of alkaloids, flavonoids, amino acids, carbohydrates, saponins, tannins, phenolic compounds, and more in the ethanolic extract, which is consistent with previous studies. Thin-layer chromatography (TLC) profiling further confirmed the presence of flavonoids, alkaloids, phenols, and tannins. LC-MS analysis identified a diverse array of compounds, including Quercetin-3-O-alpha-L-rhamnopyranoside, Vanilline, Beta-Amyrone, and various fatty acids, all of which have distinct pharmacological implications. The GC-MS analysis unveiled compounds such as Β-Sitosterol, 4-Ethylbenzoic acid, and Vitamin E, contributing to the extract's potential health benefits. HPLC analysis provided a chromatographic profile indicating distinct peaks with varying intensities and area percentages, demonstrating the complexity of the C. quadrangularis ethanolic extract. This comprehensive characterization underscores the rich phytochemical diversity and potential bioactivity of this plant, paving the way for further exploration of its therapeutic applications.