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Summary: For efficient cell entry, SARS-CoV-2 spike protein needs to be cleaved by cellular proteases. Here, we present a comprehensive protocol to assess SARS-CoV-2 spike protein cleavage in viral supernatants from SARS-CoV-2-infected cells. We also include a previous step of SARS-CoV-2 isolation from nasopharyngeal swabs of patients with COVID-19. We optimized the procedures to enhance successful viral isolation and specific spike detection. This protocol facilitates the evaluation of the role of spike mutations in spike protein processing.For complete details on the use and execution of this protocol, please refer to Escalera et al. (2022). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.