Abstract: Developing analytical methods for the chemical components of natural medicines remains a challenge due to its diversity and complexity. Miao-Fu-Zhi-Tong (MFZT) granules, an ethnic Yi herbal prescription, comprises 10 herbs and has been clinically applied for gouty arthritis (GA) therapy. Herein, a series of chemical profiling strategies including in-house library matching, molecular networking and MS/MS fragmentation behavior validation based on ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) were developed for qualitative analysis of MFZT granules. A total of 207 compounds were identified or characterized in which several rare guanidines were discovered and profiled into alkyl substituted or cyclic subtypes. Moreover, network pharmacology analysis indicated that MFZT’s anti-gout mechanism was mostly associated with the nuclear factor kappa-B (NF-κB) signaling, nucleotide oligomerization domain (NOD)-like signaling and rheumatoid arthritis pathways, along with the synergistic effect of 84 potential active compounds. In addition, a quantitative analytical method was developed to simultaneously determine the 29 potential effective components. Among them, berberine, pellodendrine, 3-feruloylquinic acid, neoastilbin, isoacteoside and chlorogenic acid derivatives at higher concentrations were considered as the chemical markers for quality control. These findings provide a holistic chemical basis for MFZT granules and will support the development of effective analytical methods for the herbal formulas of natural medicines.