Abstract only Tuberculosis (TB) remains one of the most prevalent diseases in the world, infecting one third of the world's population. The causative agent, Mycobacterium tuberculosis, relies on a plethora of lipases to maintain an infection in its host. With their central roles in infection, lipases have become a viable target for drug development, especially as drug targets for the dormant state of M. tuberculosis . This research aimed to determine the substrate specificity, biochemical properties, and potential structural information of LipN, one proposed mycobacterial lipase. Initially, wild type LipN was expressed in E. coli , purified to homogeneity, and its substrate specificity characterized against a library of 35 latent fluorophore substrates. Wild type LipN demonstrated the highest catalytic efficiency against small carbon chains with a k cat /K m over 10 5 M −1 s −1 for its best single acetyl ester substrate and maintained high activity (k cat /K m > 10 4 ) with small polar groups, including esters and oxazole rings. LipN's preference for short nonpolar substrates (three carbons or less) and polar substituents suggests its physiological role as an esterase rather than a lipase. This specificity also suggests hydrogen‐bonding capabilities within the active site of LipN. To categorize the structural factors controlling this substrate specificity, nine variants of LipN were made with substitutions in predicted binding pocket and active site residues and their relative kinetics against the top three ester substrates were characterized. Based on this analysis, the serine and histidine residues of the catalytic triad were positively identified, with 100‐fold decreases in catalytic activity upon their substitution. The aspartate residue in the catalytic triad was tentatively assigned, as multiple aspartate substituted variants showed similar reductions in catalytic efficiency. Future work will be aimed at definitely assigning the catalytic triad as well obtaining a three‐dimensional structure of LipN to confirm the structural features controlling its strict substrate specificity. Support or Funding Information Funded by NIH 1R15GM110641‐01A1