The formalin-induced paw edema test in mice and the elevated plus maze animal model were used to assess the anti-inflammatory and anxiolytic effects of Lactuca sativa L extract, respectively. Solvent evaporation was used to create solid microspheres with dispersed extract using a water-in-oil-in-water multiple emulsion system. Minitab 16 was used for optimization, and the resultant optimized microspheres were put into a gel basis. The DPPH (2,2-diphenyl1- picrylhydrazyl) test was used to assess the antioxidant activity of optimized microspheres loaded gel. Using a formalin-induced mice-paw edema test, the gel's physical characteristics, drug release kinetics, and anti-inflammatory activity were assessed. The gel's antiinflammatory capability was compared to commercially available diclofenac gel (1.16% w/w). The extract shown a dose and time dependent decrease of edema (P<0.05) at all doses, and it also demonstrated a dose dependent inhibition of anxiety level (P<0.001) in the microsphere loaded sustained release anti-inflammatory gel. Different crude extract and microsphere-loaded gel concentrations demonstrated potential antioxidant action. The anti-oxidant capability was examined using the IC50 values for ascorbic acid, the methanolic extract of L. sativa, and microsphere-loaded gel, which were 5.62 µg/mL, 6.34 µg/mL, and 8.25 µg/mL, respectively.