The use of combination drugs is considered to be a promising strategy to control complex diseases such as ischemic stroke. The detection of metabolites has been used as a versatile tool to reveal the potential mechanism of diverse diseases. In this study, the levels of 12 endogenous AAs were simultaneously determined quantitatively in the MCAO rat brain using RRLC-QQQ method. Seven AAs were chosen as the potential biomarkers, and using PLS-DA analysis, the effects of the new combination drug YQJD, which is composed of ginsenosides, berberine, and jasminoidin, on those 7 AAs were evaluated. Four AAs, glutamic acid, homocysteine, methionine, and tryptophan, which changed significantly in the YQJD-treated groups compared to the vehicle groups (P < 0.05), were identified and designated as the AAs to use to further explore the synergism of YQJD. The result of a PCA showed that the combination of these three drugs exhibits the strongest synergistic effect compared to other combination groups and that ginsenosides might play a pivotal role, especially when combined with jasminoidin. We successfully explored the synergetic mechanism of multi-component and provided a new method for evaluating the integrated effects of combination drugs in the treatment of complex diseases.