An extensive amount of knowledge on biochemistry of poly(3-hydroxyalkanoic acid) (PHA) synthesis and on its biodegradation has accumulated during the last two decades. Numerous genes encoding enzymes involved in the formation of PHA and in PHA degradation (PHA depolymerases) were cloned and characterized from many microorganisms. A large variety of methods exists for determination of PHA depolymerase activity and for preparation of the polymeric substrate (PHA). Unfortunately, results obtained with these different methods cannot be compared directly because they highly depend on the assay method applied and on the history of PHA granules preparation. In this contribution, the peculiarities, advantages, disadvantages and limitations of existing PHA depolymerase assay methods are described.