High-risk host cell serine hydrolases and other process residues have been shown to have a significant impact on the quality and safety of biologics and vaccines. In order to monitor and control process-related enzymes, particularly those present at trace levels during bioprocess development, an optimized activity-based protein profiling (ABPP) method has been developed. This method enables the identification and quantification of their activities within a single assay. This chapter provides a detailed protocol for ABPP and presents typical results obtained from the analysis of process intermediates derived from a live viral vaccine candidate.