The total iridoid glycoside extract of Lamiophlomis rotata (IGLR) has been proven to be the main pharmacological ingredient for wound healing; however, it is still unclear, which compounds in the extract are responsible for this effect. We developed and validated a mass spectrometry imaging (MSI) method to reveal the relationship between phytochemical components and the pharmacological efficacy in IGLR. Shanzhiside methyl (SM) ester, 8-O-acetylshanzhiside methyl ester (ASM), and Phlorigidoside C (PhC) have been identified as quality markers in IGLR to accelerate wound healing by MSI method. As per MSI data with UMAP manifold analysis, these compounds overlapped in the same class with endogenous metabolites (lactate, citrate and succinate). In addition, UPLC-Q/TOF-MSn coupled with multivariate analyses for skin tissues in the normal and wound groups further confirmed the results. Furthermore, MSI data from livers and kidneys revealed that SM, ASM, and PhC absorption significantly increased, whereas their elimination rates remarkably decreased in the injured group. Collectively, the overall data demonstrated that SM, ASM, and PhC selectively increased in the new granulation tissue and that the bioavailability of these three quality markers was remarkably improved in the wound group after continuous gavage for 7 days. This protocol can be applied to reveal the relationship between phytochemical components and the pharmacological efficacy of other iridoid glycoside extracts.