Lagenaria siceraria is an important medicinal plant belonging to family Cucurbitaceae. Now a day its use in Juice form is very popular. In present article, quercetin has been isolated from ethyl acetate fraction of fruit extract and confirmed by Co-TLC, UV and FTIR analysis. In addition, a simple, rapid, precise, and accurate high-performance thin-layer chromatographic (HPTLC) method has been established, validated for flavonoid quercetin. The linearity of the method was investigated in the range of 1-3.5 ng mL-1. Percentage recoveries for quercetin found to be 99.79%. The method was validated for precision, accuracy, specificity, and ruggedness. Densitometric quantification was performed at 575 nm after spraying with aluminum chloride reagent.