Nuclear accumulation of immune checkpoint PD-L1 has been associated with increased chemoresistance and cancer progression. Since nuclear PD-L1 is largely inaccessible to immunotherapiesimmunotherapies blocking cell surface PD-L1 signaling and to analytical methods measuring surface PD-L1 expression on cancer cells, it is important to develop convenient and reliable methods to measure the nuclear PD-L1 levels. Here, we describe a simple and efficient protocol that uses biochemical preparation of cytoplasmic and nuclear extracts, which are then analyzed by western blotting using PD-L1 monoclonal antibody that specifically recognizes endogenous PD-L1.