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Cellular function is critically regulated through degradation of substrates by the proteasome. To enable direct analysis of naturally cleaved proteasomal peptides under physiological conditions, we developed mass spectrometry analysis of proteolytic peptides (MAPP), a method for proteasomal footprinting that allows capture, isolation and analysis of proteasome-cleaved peptides. Application of MAPP to cancer cell lines as well as primary immune cells reveals dynamic modulation of the cellular degradome in response to various stimuli, such as pro-inflammatory signals. Further, we demonstrate analysis of minute amounts of clinical samples by studying cells from peripheral blood of patients with systemic lupus erythematosus (SLE). We find increased degradation of histones in patient immune cells, which suggests a role for aberrant proteasomal degradation in the pathophysiology of SLE. Taken together, MAPP offers a broadly applicable method to facilitate the study of the cellular degradation landscape in various cellular conditions and diseases involving changes in proteasomal degradation, including protein aggregation diseases, autoimmunity and cancer.