Chemical quenching offers a complementary approach to studying the mechanism of a flavoenzyme, supplementing the information learned from spectroscopic, structural, and computational methods. Generally, in a chemical quench experiment, an enzymatic turnover is quickly stopped at various stages with a chemical agent, and the individual reaction mixtures at each time point are analyzed for the reactants, products and any intermediates. The order by which bonds are made and broken in the reaction is indicated by the identities of the captured intermediates, and the rates of individual steps in the mechanism are determined from the amounts of various chemical species at different time points. This chapter outlines general considerations in selecting a chemical quencher of a particular enzyme-catalyzed reaction and methods for analyzing captured reaction intermediates, with a focus on flavoenzymes. The investigation of flavin-dependent thymidylate synthase is used as a case study to illustrate the concepts and workflow of quenching, isolating, and characterizing quencher-modified reaction intermediates and drawing mechanistic conclusions from the identities of these molecules. © 2019 Elsevier Inc. All rights reserved.