Dehalococcoides mccartyi (Dhc) bacterial strains expressing active reductive dehalogenase (RDase) enzymes play key roles in the transformation and detoxification of chlorinated pollutants, including chlorinated ethenes. Current state-of-the-art contaminated site monitoring regimes rely on qPCR to assess the presence of Dhc biomarker genes, however this technique alone cannot directly inform about dechlorination activity. We demonstrate the applicability of a liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS) targeted proteomics approach for the detection of Dhc biomarker proteins in groundwater contaminated with chlorinated ethenes. Targeted peptide selection was conducted in axenic cultures of Dhc strains 195, FL2, and BAV1. These experiments yielded 37 peptides from housekeeping and structural proteins (i.e., GroEL, EF-TU, rpL7/L2 and the S-layer), as well as proteins involved in the reductive dechlorination activity of Dhc strains (i.e., FdhA, TceA and BvcA). These targeted peptides can simultaneously (i.e., in a single measurement) inform about Dhc presence, general dichlorination activity, and specific reductive dechlorination degree. For example, in groundwater samples, the detection of FdhA peptides revealed active dechlorination with Dhc strain level resolution, and the detection of RDases peptides indicated specific reductive dechlorination steps. This work demonstrates the utility of the LC-MRM-MS approach for measuring reductive dechlorination biomarkers proteins in groundwater. In concert with gene-centric qPCR, environmental proteomics generates information about Dhc presence and activity, allowing better decision making for more efficiently achieving remedial goals.