<p>Lipase <i>Bacillus licheniformis</i> (LipA_{<i>B.licheniformis</i>}) is an enzyme that can be widely used in industrial applications due to its desired properties. The LipA_{<i>B.licheniformis</i>} activity is modulated by the catalytic triad as it does not have a lid and its active site is exposed under solvent. The <i>in-vitro</i> and <i>in-silico</i> analyses were conducted to identify the catalytic triad of LipA_{<i>B.licheniformis</i>} through its activity and binding affinity after mutation. Site-directed mutagenesis was performed at the hypothesized catalytic triad (Ser-77, Asp-129, and His-152), followed by the measurement of lipase activity. The binding free energies of LipA_{<i>B.licheniformis</i>} and its mutant with <i>p</i>NPL were identified through molecular docking. Based on the findings, the activity of the LipA_{<i>B.licheniformis</i>} was devoid and its binding affinity became 0&#xa0;kcal/mol after mutation induced at the hypothesized catalytic triad. The hypothesis was verified as the actual catalytic triad residues of LipA_{<i>B.licheniformis</i>} were Ser-77, Asp-129 and His-152. Mutations on these 3 residues caused loss of catalytic mechanism, thus the substrate cannot be hydrolysed. The substitution of the catalytic triad changed the LipA_{<i>B.licheniformis</i>} conformation, affecting the flexibility and stability of the active site. This study provided strong evidence to prove the identification of the actual catalytic triad of LipA_{<i>B.licheniformis</i>}, facilitating rational design to improve its performance.</p>