Bioluminescent imaging (BLI) is a foundational tool for in vivo imaging routinely used to accurately visualize cells within mouse bodies and brains. However, these probes are currently limited in their ability to image multiple targets at once as well as by their brightness. While directed evolution is commonly used to improve these enzymes, it is a slow approach and is often limited by a lack of biochemical knowledge to guide amino acid changes. Here, to rapidly generate high‐value biochemical information on the enzyme luciferase, we have developed a high‐throughput platform to categorize substrate recognition and brightness for many possible amino acid changes in a single experiment. Our approach couples screening small libraries followed by next‐generation sequencing to rapidly create sequence‐function maps. As an initial foray, we screened all mutations at three amino acid positions—A482, N229, and G246; as expected, each position showed unique patterns for their impact on function. Generally, mutations at the 482 position resulted in either inactivation of the protein or no change or slight decrease in function, while mutations at the 229 position resulted in decrease in function and a large shift towards red light emission, and mutations at the 246 position resulted in inactivation of the protein except for a handful of mutants that showed increased functionality. In addition, two mutants, N229G and G246S, were observed to have an increase in brightness with a modified luciferin substrate accompanied by a small decrease in brightness with the natural substrate, resulting in a specificity shifted towards the analogous substrate without compromised brightness. Collectively, these data illustrate that our novel, high‐throughput approach is reliable in creating diverse libraries of mutants and detecting true enrichment in function. In addition, we have identified a handful of mutants at two of the three residues investigate here that have improved brightness with non‐native substrates. This approach can be used to rapidly build a sequence‐function map at any amino acid position, facilitating future protein engineering and improving our basic biochemical understanding of this important enzyme.Support or Funding InformationFunding for this research has been provided by the Research Corporation for Science Advancement.