Objective: The aim of present work was to standardize and evaluate the antimicrobial and antioxidant activities of L. sativa leaves. Method: Physiochemical, phytochemical, and TLC technique was used for the standardization of leaves. A methanol extract of leaves was evaluated for antimicrobial activity by using agar well diffusion method and antioxidant assay by DPPH and FRAP assay. Results: Physicochemical such as loss on drying (7.19%), total ash (5.2%), acid insoluble ash (4.1%), water soluble ash (0.9%), hexane soluble (2.4%), chloroform (7.9%), methane soluble (16.2%), and water-soluble (12.6%) extractive value were identified. The bioactive secondary metabolites such as alkaloids, phenols and tannins, phytosterols and triterpenoids, saponins, anthraquinones glycosides, and cardiac glycosides were identified. RP-TLC and normal TLC studies of the methanol extract were carried out and the observations will help in identification of crude powder. Total phenolic and flavonoid content of the methanol extract was found to be 53.64 ± 0.27mg GAE/g and 16.55 ± 0.49mg QE/g using Folin-Ciocalteu and AlCl3 colorimetric method respectively. The antibacterial activity of the extract was exhibited against S. aureus, S. aureus (CI), B. subtilis, and E. coli at concentration 20mg/ml. The zone of inhibition ranged from 8.2-13.8 mm, MIC's ranged from 6.25 to 25 mg/ml and MBC of 50 and ˂50 mg/ml. In the DPPH assay, the IC50 value of methanol extract and ascorbic acid were 210.57 µg/ml and 31.92 µg/ml respectively. Conclusions: These results clearly indicate that L. sativa is to be a natural source of antioxidant and it could be alternative antibiotics. Keywords: Lactuca sativa, leaves, standardization, antimicrobial, antioxidants.