OBJECTIVE： To establish a method for the determination of related substances in Timolol maleate eye drops， and to conduct qualitative analysis of its degradation impurities. METHODS： HPLC method was adopted for determination of related substance. The determination was performed on ACES C18 column with mobile phase consisted of the mixed solution of methanol and 4.32 g/L sodium octane sulfonate （pH adjusted to 3.0 with glacial acetic acid）（50 ∶ 50，V/V）-methanol （gradient elution） at flow rate of 1.0 mL/min. The detection wavelength was set at 295 nm， and column temperature was 30 ℃. The sample size was 20 μL. Preparative LC method was used to prepare pure impurities.  The determination was performed on YMC-PACK ODS-A column with mobile phase consisted of methanol-0.01％ trifluoroacetic acid （40 ∶ 60，V/V） and methanol-0.01％ trifluoroacetic acid （20 ∶ 80，V/V） at flow rate of 8 mL/min. The detection wavelength was set at 295 nm， and column temperature was 20 ℃. The sample size was 0.8 mL. LC-MS/MS method was adopted. The determination was performed on Waters ACQUITY UPLC BEH C18 column with mobile phase consisted of methanol-5 mmol/L ammonium formate （0.05％ formic acid） （gradient elution） at flow rate of 0.2 mL/min. The column temperature was 20 ℃. The sample size was 2 μL. The electrospray ionization source was conducted， detecting patient ion by MS full scan and fragment ion by daughter ion scan. The structure was confirmed and speculated by 1H-NMR and 13C-NMR. RESULTS： The linear range of timolol maleate was 0.501-10.02 μg/mL （r＝0.999 9）. The limit of quantitation was 3.012 ng， and the limit of detection was 1.004 ng. RSD of precision test was 0.2％. RSDs of stability and reproducibility tests were all lower than 5％. The structures of impurityⅠ， Ⅱ and Ⅲ were confirmed， and those of impurity Ⅳ and Ⅴ were speculated. CONCLUSIONS： The method is rapid， accurate and specific. It can be used for the determination of related substance in Timolol maleate eye drops.