Rooibos (Aspalathus linearis) is a rich source of polyphenols and used to make a mild-tasting tea containing no caffeine, is low in tannins compared to green or black teas, and has antioxidant and antimutagenic/antitumoral properties. In vivo results show that rooibos has beneficial effects upon the lipid profile by decreasing serum triglycerides and cholesterol. In this sense, we have developed a simple and rapid method to separate and characterize simultaneously the polyphenolic compounds in aqueous and ethanolic rooibos extracts using high-performance liquid chromatography coupled to electrospray ionization time-of-flight mass spectrometry (HPLC-ESI-TOF-MS) and ion trap multiple mass spectrometry (HPLC-ESI-IT-MS(2)). The phenolic compounds were separated on a C(18) column (4.6 × 150 mm, 1.8 μm) with 1% formic acid in water/acetonitrile 90:10 v/v and acetonitrile as mobile phases. The accuracy mass data generated by TOF-MS together with the fragmentation pattern obtained by IT-MS(2) experiments confirmed the presence of 25 and 30 phenolic compounds in the aqueous and ethanolic extracts, respectively.