Ubiquitination is an essential protein modification that influences multiple eukaryotic processes，such as substrate degradation, pathway alterations. However, because of the interaction between the substrate and other proteins, it is difficult to detect the exact ubiquitination level of a specific protein using immunoprecipitation and western blot method in a substrate-overexpression cell line. In our nature paper, we used a denaturing conditions to eliminate proteins conjugated with haemagglutinin-tagged PD-1. By using this protocol, we can detect the exact ubiquitination level of PD-1 with western blot analysis. After cells have been collected, the described protocol can be completed in 2–3 d.