High-performance liquid chromatography (HPLC) coupled with electrospray tandem mass spectrometry (ESI-MS-MS) and diode array detection (DAD) was used to identify and simultaneously determine eight major ingredients in Radix Tinosporae. The assay was performed on a Diamonsil C18 analytical column with a gradient solvent system of A (water containing 0.2% formic acid, 20 mM ammonium acetate) and B (methanol/acetonitrile = 1/1, v/v). The 217, 248, 270 and 347 nm, respectively, were chosen as the monitoring wavelengths to determine four structural types of components, say columbin, phytoecdysteroids (including 20-hydroxyecdysone, 2-deoxy-20-hydroxyecdysone 3-O-β-d-glucopyranoside and 2-deoxy-20-hydroxyecdysone), menisperine and protoberberine alkaloids (including columbamine, jatrorrhizine and palmatine). This method was validated in respect to precision, repeatability and accuracy, and was successfully applied to quantify the eight components in 39 batches of R. Tinosporae for quality control purpose. The results indicated that the proposed method could be readily utilized as a quality control method for traditional Chinese medicine (TCM).