Cobratide is a peptide drug extracted from the venom of Chinese cobra, and has been widely used in the clinical treatment of chronic, intractable and persistent pain. In a recent study, it was reported that it has the potential to treat COVID-19. In order to control the quality of commercial cobratide drugs, a protocol was established for the separation, identification and quantification of cobratide and its associated impurities, in which sheathless capillary electrophoresis-mass spectrometry (CE-MS) was used for identification and a rapid capillary electrophoresis-ultraviolet-visible detector (CE-UV) method was developed for accurate quantification. Separation conditions that affect the resolution and MS intensities of cobratide and its associated impurities were investigated, including pH value, concentration of background electrolyte (BGE), ratio of organic additive and sample solution. The optimized CE conditions (BGE: 50 mM NH4Ac, pH 4.0; sample solution: deionized water) were used for both sheathless CE-MS and CE-UV methods. Three associated impurities were separated and identified for the first time by sheathless CE-MS. Then, a rapid CE-UV method was validated and used for accurate quantification of cobratide and its associated impurities. The CE-UV method showed good linearity between concentration and corrected peak area of cobratide in the concentration range of 5.36-536.30 μg mL-1. The limit of quantification of the CE-UV method was 4.16 μg mL-1. The relative standard deviations of migration time were less than 1% for both intra-day and inter-day experiments, and those of corrected peak area were less than 5%. Finally, different cobratide drugs were analyzed to evaluate the batch-to-batch consistency. This established protocol combining sheathless CE-MS and CE-UV methods would provide useful information for both quality control and process analysis of peptide drugs.