Directed evolution is a well-established and powerful tool for enzyme engineering, which consists of iterative rounds of creating and screening a library of variants. In many cases, the ability to characterize these variants in high-throughput remains a bottleneck. In addition, profiling of desired candidates becomes even more challenging when engineering multiple enzymes in a biochemical pathway. In this chapter, we describe a label-free, high-throughput method for the engineering of multistep enzymatic reactions in bacterial colonies via optically guided matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry (MS). This method is able to detect products, reactants, and byproducts with high sensitivity and accuracy. We demonstrate its effectiveness in two applications related to natural product biosynthesis, including facile creation of analog of the peptidic antibiotic plantazolicin and rapid profiling of congeners of rhamnolipid. Computational algorithms were developed to process and visualize the resulting mass spectral data sets. In both cases, improved MS acquisition efficiency and information-rich insights were obtained through this technique on large populations of colonies at a rate of 1–2.5 s per colony. This method should be generally applicable to high-throughput phenotyping of microbial libraries from a wide range of enzymatic reactions.