An efficient method was developed and established for the rapid analysis of a traditional Uighur medicinal prescription, Yizhihao granule, by liquid chromatography-Quadrupole time-of-flight tandem mass spectrometry. HPLC was performed with a Poroshell 120-EC18 column with 2.7 µm particle size using A gradient elution system. A quadrupole time-of-flight analyzer was used for the determination of accurate mass of the protonated or deprotonated molecules and fragment ions. Sixty-six constituents including amino acids, organic acids (aliphatic acids, benzoic acid derivatives, and chlorogenic acids), alkaloids (indole and quinazolinone type), flavonoids (C,O-glycoside, C-glycosides, O-glycosides, and aglycone), sesquiterpenoids (rupestonic acid derivatives), and other compounds were identified or tentatively characterized in Yizhihao granule as a presented application of prescription according to the accurate mass, MS/MS data, and relevant literature. Additionally, the fragmentation pathways of two rupestonic acid derivatives were proposed. The relationship between structure features and fragmentation patterns were investigated thoroughly and applied in the on-line analysis to deduce the composition of traditional Uighur herbal prescription. The present method and results should be beneficial in the development of traditional Uighur herbal prescription.