The fruit of Ziziphus jujuba Mill., known as Hongzao (or Hong-Zao) in Chinese and cultivated in China for more than 4000 years, has shown to have hepatoprotective property. In previous study, we have isolated and identified 27 known compounds from Z. jujuba fruits, which demonstrated anti-tumor activity. In this study, a high-performance liquid chromatography-diode-array detection-mass spectrometry (HPLC-DAD-MS) method was successfully applied to the simultaneous characterization and quantitation of 18 constituents in 28 Z. jujuba samples, comprised of 12 cultivars from different regions in China, by comparing their HPLC retention times, MS spectra, UV spectra, and NMR data with those of reference compounds. The quantitative method was validated with excellent linearity (R2 > 0.999 1), preferable intra- and inter-day precisions (RSD < 2.78%), and good recoveries (94.96%–102.65%). The content variation of 18 compounds was analyzed by a chemometric method (hierarchical cluster analysis). In addition, these constituents showed protection against carbon tetrachloride (CCl4) intoxicated HepG2 cell lines by decreasing lactic dehydrogenase (LDH) levels. Results in this study illustrated that the content of all 18 compounds examined has significant difference and variation among cultivars and extracts. The proposed method can serve as a prerequisite for quality control of bioactive compounds in Z. jujuba products.