A novel quantitative antioxidant activity assay based on cellular metabolomics, named cellular metabolomics antioxidant activity (CMAA) assay, was established and applied in this paper. CMAA assay evaluates antioxidant activity by considering the change rules of the cellular metabolites of different treatment groups of human liver carcinoma cell line HepG2 cells subjected to oxidative stress. This assay considers cellular metabolites as the main analytical index, principal components analysis (PCA), and orthogonal projections to latent structures discriminant analysis (OPLS-DA) as the main multi- and megavariate data analysis method and vitamin E (VE) as a standard substance to quantify antioxidant activities. The antioxidant activities of flavonoids extracted from Cynomorium songaricum Rupr. (CS) were measured by the CMAA assay. The results showed that the antioxidant activity of flavonoids extracted from CS was successfully evaluated using the CMAA assay, and the antioxidant activity of flavonoids extracted from CS was about 1/5 of the same concentration of VE. It indicated that the proposed approach, as an effective tool for evaluating the antioxidant capacity, was feasible.