Rhomboid proteases were discovered almost 15 years ago and are structurally the best characterized intramembrane proteases. Apart from the general serine protease inhibitor 3,4-dichloro-isocoumarin (DCI) and a few crystal structures of the Escherichia coli rhomboid GlpG with other inhibitors, there is surprisingly little information about inhibitors of rhomboids from other species, probably because of a lack of general methods to measure inhibition against different rhomboid species. We here present activity-based protein profiling (ABPP) as a general method to screen rhomboids for their activity and inhibition. Using ABPP, we compare the inhibitory capacity of 50 small molecules against 13 different rhomboids. We find one new pan rhomboid inhibitor and several inhibitors that display selectivity. We also demonstrate that inhibition profile and sequence similarity of rhomboids are not related, which suggests that related rhomboids may be selectively inhibited. Finally, by making use of the here discovered inhibitors, we were able to show that two bacterial rhomboids autoprocess themselves in their N-terminal part.