Liquid chromatography coupled to electrospray ionization tandem mass spectrometry (MSn) is used for the analysis of flavonoids in heartsease (Viola tricolor L.). Our data suggested that the two main flavonoid components were violanthin (6-C-glucosyl-8-C-rhamnosyl apigenin) and rutin (3-O-rutinosyl quercetin). The identification of rutin was confirmed by comparing its retention time, UV spectrum, molecular mass, and fragmentation pattern with the reference standard. In this paper, we also report on the quantitative analysis of rutin by high-performance liquid chromatography. According to our results, heartsease herb contained 420+/-1.17 microg/g rutin.