GC/MS coupled with multivariate statistical analysis was performed to identify marker metabolites in serum of mice after healing ovalbumin- (OVA-) induced asthma using Opuntia humifusa. Principal component analysis (PCA) score plot showed separation among groups, with metabolite profiles of serum showing differences according to various treatments for the asthma murine model. Levels of stearic acid and arachidic acid were significantly lower in the serum from OVA-induced group than those from the control group. Dexamethasone treatment group was characterized by higher serum levels of urea, myristic acid, and palmitic acid along with lower levels of aspartic acid compared to OVA-induced group. O. humifusa treatment mice groups showed dose-proportional higher levels of urea and glycerol than OVA-induced group. These results highlight that GC/MS-based metabolomics is a powerful technique for identifying molecular markers of asthma.