4‐Substitution on proline directly impacts protein main chain conformational preferences. The structural effects of N‐acyl substitution and of 4‐substitution were examined by NMR spectroscopy and X‐ray crystallography on minimal molecules with a proline 4S‐nitrobenzoate. The effects of N‐acyl substitution on conformation were attenuated in the 4S‐nitrobenzoate context, due to the minimal role of the n→π* interaction in stabilizing extended conformations. By X‐ray crystallography, an extended conformation was observed for most molecules. The formyl derivative adopted a δ conformation that is observed at the i+2 position of β‐turns. Computational analysis indicated that the structures observed crystallographically represent the inherent conformational preferences of 4S‐substituted prolines with electron‐withdrawing 4‐position substituents. The divergent conformational preferences of 4R‐ and 4S‐substituted prolines suggest their wider structure‐specific application in molecular design. In particular, the proline endo ring pucker favored by 4S‐substituted prolines uniquely promotes the δ conformation [(ϕ, ψ) ≈(−80°, 0°)] found in β‐turns. In contrast to other acyl capping groups, the pivaloyl group strongly promoted trans amide bond and polyproline II helix conformation, with a close n→π* interaction in the crystalline state, despite the endo ring pucker, suggesting its special capabilities in promoting compact conformations in ϕ due to its strongly electron‐donating character.