Rhomboid proteases form a paradigm for intramembrane proteolysis and have been implicated in several human diseases. However, their study is hampered by difficulties in solubilization and purification. We here report on the use of polymers composed of maleic acid and either diisobutylene or styrene for solubilization of rhomboid proteases in lipid nanodiscs, which proceeds with up to 48% efficiency. We show that the activity of rhomboids in lipid nanodiscs is closer to that in the native membrane than rhomboids in detergent. Moreover, a rhomboid that was proteolytically unstable in detergent turned out to be stable in lipid nanodiscs, underlining the benefit of using these polymer-stabilized nanodiscs. The systems are also compatible with the use of activity-based probes and can be used for small molecule inhibitor screening, allowing several downstream applications.