Abstract: Baicalin eye drops have broad clinical application prospects in the treatment of ocular diseases. However, there is still a lack of qualitative and quantitative research on the related substances in them. This study first established an analytical method for related substances in baicalin eye drops, providing a scientific basis for product development and quality control. Through LC-Q/TOF-MS/MS, seven related substances were preliminarily identified in baicalin eye drops, namely scutellarin, oroxylin A-7-O-glucuronide, oroxin A, quercetin-7-O-glucuronide/quercetin 3-O-glucuronide, chrysin-7-O-glucuronide, baicalein, and scutellarein. The quantitative analysis of oroxin A, scutellarin and oroxylin A-7-O-glucuronide was carried out by HPLC. The chromatographic column was the NanoChrom AR C18 column (250 mm × 4.6 mm, 5 μm). The mobile phase was 0.1% phosphoric acid aqueous solution (A)-acetonitrile (B), with gradient elution. The column temperature was 20 ℃, the flow rate was 0.8 mL·min^-1, and the detection wavelength was 254 nm. The established method has strong specificity, high sensitivity, and good precision. In this study, a method for the qualitative and quantitative analysis of related substances in baicalin eye drops was established, providing a basis for ensuring their quality.