A continuous spectrophotometric assay for the screening of PHB depolymerase activity in microtiter plates was developed. We evaluated crystalline PHB in the suspension and coated it with the addition of a pH indicator to detect the breakage of the ester bond by proton titration. The reaction rate and the concentration of the recombinant PhaZ1 from Paucimonas lemoignei PHB depolymerase presented a linear correlation. A comparison of the proposed method with the turbidimetric method adapted to the microtiter plates revealed that the use of indicators increases the response signal by at least 5-fold, resulting in increased sensitivity and better signal-to-noise ratio. Furthermore, the proposed method offers a wide range of pH from 5.0 to 9.2 by using different buffer-indicator pairs and was employed for the screening of PHB-depolymerase activity on 140 bacterial strains isolated from Lake Chapala. Eleven strains were positive for PHB-depolymerase activity, which were ACSLRF-27, ACPLRF-6, and ACPLRF-5 (16S rRNA sequence alignment revealed 99-100% similarity with Actinomadura geliboluensis strain A8036, Streptomyces cavourensis strain NRRL 2740, and Streptomyces coelicolor strain DSM 40233, respectively); these that showed the highest activities. In conclusion, the method was successfully applied for finding new strains and for quantifying the PHB depolymerases activity with crystalline PHB.