A majority of thousands of intracellular mammalian proteins are recognized by proteasome only being conjugated with ubiquitin (Ub), representing a universal degradation signal operated by the ubiquitination system. Ub‐independent proteasome targeting is rationalized by the existence of 2 types of direct proteasome signals (DPSs), specific amino acid sequences or post‐translational modifications, which are recognized by proteasome regulatory subunits. Historically, the first type was shown to exist in ornithine decarboxylase, whereas acetylation of core histones recently was reported as a second type of DPS. Here we declare a third type, representing charge‐mediated DPS. This discovered DPS may be classified as a monopartite composition‐ but not sequence‐dependent element of ∼70 Å in length enriched in basic and flexible amino acids. This type of degradation signal, which may be provided by cationic chemicals, is most efficiently engaged by proteasomes capped with regulator (REG)α or REGγ in an ATP‐independent manner. Taken together, our findings suggest a novel modality of proteasome‐substrate interrelation bypassing ubiquitination.—Kudriaeva, A., Kuzina, E. S., Zubenko, O., Smirnov, I. V., Belogurov, A. Charge‐mediated proteasome targeting. FASEB J. 33, 6852–6866 (2019). http://www.fasebj.org