The observation that tumour cells are more sensitive to pharmacological inhibition of the proteasome than normal cells has led to the development of the proteasome inhibitor bortezomib. To date, this is the only proteasome inhibitor that has been approved for clinical use. The clinical success of bortezomib, combined with the occurrence of adverse effects and the development of clinical resistance against this compound, has initiated the development of a broad range of second-generation proteasome inhibitors as well as of assays that can be used to establish a relationship between the extent and type of proteasome inhibition and the effectiveness of a particular drug. In the present paper, we discuss new strategies that may be used in the future to overcome drug resistance and to broaden the use of proteasome inhibitors for the treatment of both cancer and infectious and autoimmune disease.