Biocatalysis in ionic liquids (ILs) has received tremendous attention in the manufacture of biodiesel, sugar esters, and pharmaceuticals. However, hydrophilic IL interaction with enzymes often leads to decreased activity or even inactivation. In this report, we prove that intrinsic lipase stability and the maintenance of hydration shells of Bacillus subtilis Lipase A (BSLA) are two synergistic design principles to maintain enzymatic activity in ILs. After this in silicoWhen screening nine advantageous amino acid positions according to the CompassR rule (172 variants in total), we rationally designed two variants to be constructed by site-directed mutagenesis and three libraries by site-directed mutagenesis. With a minimal amount of testing, we identified three all-round variants against four [BMIM] -based ILs. Remarkably, the variant M1a F17S / V54K / D64N / D91E / G155N had a 6.7 times higher resistance to 40% (v / v) [BMIM] Cl, 5.6 fold in 80% (v / v) [BMIM] Br, 5.0-fold in 30% (v / v) [BMIM] [TfO] and 2.7 times in 10% (v / v) [BMIM] I compared to wild type BSLA while showing a 1.9 fold improvement in specific activity. The computer-aided analysis of the molecular dynamics and the thermodynamic stability analysis of the variants resulted in the molecular basis for the resistant variants M1a and M1b as a synergistic improvement in protein stability (ΔΔ)Gwrinkles in the range of -4.26 to -4.80 kcal mol−1) and increased hydration cups around the substitutions in the four ILs (up to 1.7 fold). These design principles and the molecular knowledge gained not only open up the possibility for direct experimenters to rationally design promising IL-resistant enzymes, but also offer new insights into enzymatic catalysis in ILs.