The successful implementation of immune checkpoint inhibitors (ICIs) immunotherapy into clinical routine has underlined the importance of immunotherapy for the treatment of cancer. Nevertheless, benefits from ICI monotherapy remain limited to a subset of patients. The induction of immunogenic cell death (ICD) can prime tumors for subsequent ICI via the onset of adaptive immune responses leading to an infiltration of cytotoxic T lymphocytes. The nuclear and cellular nuclear release of high mobility group box 1 (HMGB1) is one of the hallmarks of ICD. Binding of HMGB1 to Toll-like receptor 4 (TLR4) expressed on dendritic cells plays a pivotal role in stimulating their maturation and antigen presentation, facilitating the onset of adaptive immunity. Here we describe microscopic assessments of HMGB1 release that can be applied to the screening of chemical compound libraries for novel ICD inducing agents. Thus, quantitative measurement of HMGB1 release kinetics can be useful for the discovery of new immuno-oncology drugs.