The purpose of this study was to develop reversed-phase HPLC method for the simultaneous determination of the flavonoids from the leaves of Nelumbo nucifera, which have been known to exhibit antioxidant, anti-HIV, antihyperlipidemic and antiobesity effects. HPLC separation was achieved on $C_{18}$ column using gradient elution with mobile phase consisting of acetonitrile-water containing 0.1% formic acid. The separated peaks were identified by electrospray ionization mass spectrometry. The HPLC method was validated and applied for the simultaneous determination of the bioactive flavonoids from the leaves of Nelumbo nucifera. In the methanol extract, six flavonoids, quercetin, rutin, quercetin 3-O-$eta$-D-galactopyranoside (Qc-3-Gal), quercetin 3-O-$eta$-D-glucopyranoside (Qc-3-Glc), quercetin 3-O-$eta$-D-glucuronide (Qc-3-Gln) and quercetin 3-O-$alpha$-arabinopyranosyl-($1{ ightarrow}2$)-$eta$-galactopyranoside (Qc-3-AraGal), were identified. Among them, Qc-3-Glc and Qc-3-Gln were found to be major component in the methanol extract of Nelumbo nucifera leaves.