The purpose of this research is to recognize the active antitumor components from the mixed pair extract of Aconiti Lateralis Radix Praeparata (Fuzi in Chinese) and Glycyrrhizae Radix et Rhizoma (Gancao in Chinese) using chemometrics and mean impact value (MIV) methods. Firstly, 30 common components of 31 different samples were analyzed quantitatively and qualitatively by HPLC-UV and UPLC-Q-TOF tandem mass spectrometry, respectively. Meanwhile, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays were used to test the inhibition activities of the 31 different samples against HeLa cells. Then a back propagation (BP) neural network, support vector regression (SVR), and two optimization algorithms – genetic algorithm (GA) and particle swarm optimization (PSO) – were applied to construct composition–activity relationship (CAR) models for the Fuzi–Gancao extract. Based on the optimal CAR model, the MIV was introduced to evaluate the contribution of each individual component to the anticancer efficacy of the extract. Results indicated that the SVR-PSO model best depicted the complex relationship between the chemical composition and the inhibition effect of a Fuzi–Gancao extract. The 30 common components were ranked by their absolute MIVs, and the top 8, which corresponded to peaks 17, 25, 22, 13, 23, 28, 5, and 7 in the chromatogram, were tentatively deemed to be the main antitumor components. The integrated strategy shows a novel and efficient approach to understanding the potential contributions of components from complicated herbal medicines, and the identified results suggest certain directions for screening and research into new antitumor drugs.