Herein, we report that the conformational preference of phenyl acetate is governed by steric effect and n→π* interaction. Conformation-specific electronic and IR spectroscopy combined with quantum chemistry calculations confirm the presence of only the cis conformer of phenyl acetate in the experiment. The cis conformer of phenyl acetate has n→π* interaction between the lone-pair electrons on the carbonyl oxygen atom and the π* orbitals of the phenyl group. The n→π* interaction is absent in the trans conformer which has additional steric repulsion between the methyl group and phenyl ring. The trans conformer is higher in energy than the cis conformer by ≈3 kcal mol-1 . We have found the effect of methyl substitution on the strength of the n→π* interaction, steric repulsion, and hyperconjugation in phenyl acetate. The red-shift observed in the cis conformer of phenyl acetate with respect to the trans conformer is affected due to the influence of the methyl substituent on the strength of the n→π* interaction as well as hyperconjugation. The present result demonstrates that the introduction of a bulkier substituent can induce steric as well as electronic control to reduce conformational heterogeneity of a molecular system. Understanding the effect of bulkier substituents to promote defined conformations having specific non-covalent interactions may have implication in better perception of the optimum structure and function of biomolecules as well as recognition of drugs by biomolecules.