A stage of drug discovery during which a compound demonstrated to have antibacterial properties (a hit) is iteratively modified to improve its efficacy and other properties.

Typically, multiple chemical variants of a hit compound are synthesized (hit expansion). Studies of these compounds help to identify key structural features of the compound associated with binding to molecular targets and efficacy (structure–activity relationships).

Chemical modifications are also introduced to improve its drug-like properties, such as stability, membrane permeability, cytotoxicity and specificity.

This stage ends with one or more optimized lead compounds that are extensively evaluated for efficacy and safety in biochemical and cellular assays and in animal studies.