According to the theory of traditional Chinese medicine, the kidney is regarded as governing the bones and dominating the storage of essence. Gushudan (GSD) is a traditional Chinese medicine prescription that has the effects of strengthening bone and nourishing the kidney. However, the mechanism of action of GSD in preventing postmenopausal osteoporosis (PMOP) rats based on the 'kidney-bone' axis remains to be further systematically investigated. In this study, an integrated kidney metabolomics method based on three MS detection modes of UHPLC-HRMS, UHPLC-MS/MS and AFADESI-MSI was developed to reveal the protective mechanism of GSD in PMOP rats. Firstly, the non-targeted metabolomics was investigated to comprehensively explore the metabolic changes in the kidneys of PMOP rats based on the UHPLC-Q-Orbitrap HRMS. Subsequently, UHPLC-MS/MS targeted metabolomics and Mass Spectrometry Imaging (MSI) techniques were combined to elucidate the preventive mechanism of GSD on PMOP through branched-chain amino acid (BCAA) metabolism. The results of the non-targeted metabolomics demonstrated that GSD significantly modulated the levels of 67 potential biomarkers, including leucine and valine, which are primarily involved in amino acid metabolism. Specifically, BCAA metabolism is notably enriched in amino acid metabolism. Compared to the control group, it was found that the levels of BCAAs were decreased and α-branched-chain keto acids (BCKAs) were increased in the model groups in the targeted metabolomics study. Moreover, MSI results showed that the changes in BCAAs content were mainly concentrated in the renal cortex. This finding confirmed the metabolic disorders of BCAA in the renal cortex of PMOP rats, and that GSD had a significant regulatory effect on this disorder. In conclusion, this study integrated three mass spectrometry techniques that validate and complement each other to revealed the anti-osteoporostic mechanism of GSD in PMOP rats and to elucidate the modern scientific connotation of the 'kidney-bone' axis based on the BCAA metabolism.