Introduction: Salvia hispanica L. is an annual herbaceous plant commonly known as "Chia", native of southern Mexico and northern Guatemala. The aim of this study is isolation, identification of secondary metabolites and evaluation of biological activities of ethyl acetate fraction of Salvia hispanica L. aerial parts. Methods: Air dried powdered of Salvia hispanica L. aerial parts was extracted by maceration and fractionated using light petroleum, dichloromethane and ethyl acetate solvents. Ethyl acetate fraction was subjected to column and thin layer chromatography for isolation of secondary metabolites that are characterized by UV-Vis, FT-IR, EI-MS, 1D and 2D NMR spectral analyses. UPLC-ESI-MS/MS technique was used on the same fraction. In-vitro biological evaluation of the fraction carried out for anti-oxidant activity using DPPH assay, anti-obesityactivity using pancreatic lipase inhibitory assay,anti-diabetic activity usingα -amylase inhibition assay andanti-cancer activities usingcell viability assay. Results: Six compounds were isolated including 1,2,4,5 tetrahydroxy benzene (1), leucantho flavone (2), rhamnetin (3), apigenin-7-O-β- D-glucoside(4), rosmarinic acid (5) and kaempferol-7-O-β-D-glucoside (6). The identification of thirty seven compounds byUPLC-ESI-MS/MS analysis. A strong DPPH scavenging activity with IC50 13.11compared to ascorbic acid, anti-obesity activity with IC50 59.3 compared to orlistate, anti-diabetic activity with IC50 95.2 compared to acarbose. High cytotoxic activity against lung carcinoma, colon carcinoma and moderately cytotoxic activity against prostate carcinoma cell lines. Conclusions: Salvia hispanica L. is a strong antioxidant and anti-carcinogenic against lung and colon cancer.