Halogenated contaminants, particularly brominated flame retardants, disrupt circulating levels of thyroid hormones (THs), potentially affecting growth and development. Disruption may be mediated by impacts on deiodinase (DI) activity, which regulate the levels of active hormones available to bind to nuclear receptors. The goal of this study was to develop a mass spectrometry-based method for measuring the activity of DIs in human liver microsomes and to examine the effect of halogenated phenolic contaminants on DI activity. Thyroxine (T4) and reverse triiodothyronine (rT3) deiodination kinetics were measured by incubating pooled human liver microsomes with T4 or rT3 and monitoring the production of T3, rT3, 3,3'-diiodothyronine, and 3-monoiodothyronine by liquid chromatography tandem mass spectrometry. Using this method, we examined the effects of several halogenated contaminants, including 2,2',4,4',5-pentabromodiphenyl ether (BDE 99), several hydroxylated polybrominated diphenyl ethers (OH-BDEs), tribromophenol, tetrabromobisphenol A, and triclosan, on DI activity. The Michaelis constants (K(M)) of rT3 and T4 deiodination were determined to be 3.2 ± 0.7 and 17.3 ± 2.3μM. The V(max) was 160 ± 5.8 and 2.8 ± 0.10 pmol/min.mg protein, respectively. All studied contaminants inhibited DI activity in a dose-response manner, with the exception of BDE 99 and two OH-BDEs. 5'-Hydroxy 2,2',4,4',5-pentabromodiphenyl ether was found to be the most potent inhibitor of DI activity, and phenolic structures containing iodine were generally more potent inhibitors of DI activity relative to brominated, chlorinated, and fluorinated analogues. This study suggests that some halogenated phenolics, including current use compounds such as plastic monomers, flame retardants, and their metabolites, may disrupt TH homeostasis through the inhibition of DI activity in vivo.