Passiflora species have been widely used in folk medicine as tranquilizers, and previous pharmacological studies have reported sedative activity for P. quadrangularis L., Passifloraceae, leaf extracts. The aim of this work was to contribute to the standardization of P. quadrangularis leaf extract with sedative activity. For this purpose, the extraction of total flavonoids was optimized, evaluating variables such as drug-solvent ratio, extraction solvents and extraction time, using Response Surface Methodology. The stability of total and individual flavonoids on the optimized extract of P. quadrangularis leaves under stress conditions was also evaluated. Sedative activity was verified by the ethyl ether-induced hypnosis test in Swiss ICR mice. Based on the results, the highest concentration of total flavonoids was obtained at a drug-solvent ratio of 1:15 (w:v), extraction solvent EtOH:H2O (1:1, v/v) and percolation time of 48 h. Regarding stability under stress conditions, it was found that the flavonoids from the optimized extract are photostable, and practically stable under neutral hydrolysis and oxidation, but labile by acid and basic hydrolysis, with the main degradation products being identified. Finally, it was demonstrated that the optimized extract improves the sedative effect when compared to previously evaluated extract in the ethyl ether-induced hypnosis test.