Laportea bulbifera(Siebold & Zucc.) Wedd., Urticaceae, is a medicinal herb customarily used by the Miao people in China, for the treatment of rheumatism, traumatic injury, and other symptoms. The aim of this study was to develop and validate a sensitive and reliable high-performance liquid chromatography-tandem mass spectrometry method for the simultaneous quantitation of five constituents, including neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, and kaempferol-3-O-rutinoside, of L. bulbiferaextract in biological samples, and to apply the method to a pharmacokinetic study. The validated method was successfully applied to a pharmacokinetic study in rats with oral administration of L. bulbiferaextract at a single dose of 8.3 g kg−1. The five constituents were detected and their pharmacokinetic parameters were calculated. The established method was rapid and sensitive and could be used for the pharmacokinetic study of L. bulbiferaextract.