Lysosomal acid lipase (LAL), encoded by the LIPA gene, is essential for lipid metabolism, and its deficiency causes Wolman disease and cholesteryl ester storage disease. Understanding the structural determinants of LAL stability and function is crucial for protein engineering and therapeutic development. We integrated evolutionary analysis with computational structural biology to identify and characterize functionally important residues in human LAL. Using codon-based models with Bayesian empirical Bayes methods on 11 primate orthologs, we identified two positively selected sites, L197 and C232 (cutoff posterior probability ≥ 95%). Site C232, located in the functionally critical lid region controlling substrate access, was selected for comprehensive in silico mutagenesis. FoldX stability calculations across all 19 amino acid substitutions revealed six stabilizing mutations, with C232L showing the strongest effect (ΔΔG = -3.06 kcal/mol). Molecular docking with the lipase inhibitor orlistat showed enhanced binding affinity for C232L (-7.40 kcal/mol) compared to wild type (-6.47 kcal/mol). Molecular dynamics simulations (100 ns) demonstrated that C232L maintained superior structural stability with reduced RMSD (0.28-0.35 nm vs. 0.45-0.60 nm for wild type) and decreased lid region flexibility. Principal component analysis revealed more constrained conformational sampling, while free energy landscape analysis indicated a single, well-defined energy minimum for C232L. MMPBSA calculations confirmed stronger binding energetics (-199.45 ± 15.42 kJ/mol) driven by enhanced van der Waals and electrostatic interactions. Our results demonstrate that positive selection at C232 has tuned lid dynamics to optimize orlistat binding. The C232L substitution represents a promising target for experimental protein engineering to enhance LAL stability for therapeutic applications.