Activity-based protein profiling (ABPP) is a chemoproteomic technique that uses functionalized covalent enzyme inhibitors known as activity-based probes (ABPs) to detect, quantify, and identify active enzymes in a biological sample. Here, we describe experimental protocols for profiling of enzyme activities in bacterial cultures using both fluorescent and biotinylated ABPs that allow for the gel-based detection and resolution of target enzymes, or their enrichment and mass spectrometry-based identification, respectively. These protocols have been optimized for profiling of serine hydrolases using fluorophosphonate probes in both Gram-positive and Gram-negative bacteria, and they can easily be adapted for the application of probes targeting other enzyme families of interest in bacteria under diverse growth conditions.