In microbial biodegradation assays, the detection of bacterial growth in water-based plastic dispersions can be difficult to measure using traditional methods because of the turbidity of culture media and the formation of flocculi. Here, we present a protocol for the detection of bacterial growth in Impranil®DLN, a polyester polyurethane (PU) water-based dispersion. By measuring bacterial metabolic activity, as an indicator of cell viability with the water-soluble 2, 3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) salt. Viable growing cells, i.e., those cells that can utilize PU as a carbon source, will reduce the yellow-colored XTT to a water-soluble orange formazan by the action of dehydrogenase enzymes of the respiratory chain. For the standardization of the protocol, we used Pseudomonas putida KT2440 and Escherichia coli BL21 strains as positive and negative controls, respectively. We determined the metabolic activity of the strains grown with citrate or both citrate and impranil as carbon sources. P. putida KT2440 showed higher XTT-detected metabolic activity in the presence of PU than when it was grown only with citrate, indicating that the strain also used PU as a carbon source. In contrast, the negative control did not show differences in metabolic activity between the growth conditions. Our protocol can be adapted to different bacterial strains and culture media.