The formation of pyrene metabolites by the terrestrial isopod Porcellio scaber (Latr.) was studied following exposure for 13 days to 100 μg/g pyrene in its food. An alkaline hydrolysis method with subsequent liquid-liquid extraction was developed to determine the metabolite content, especially 1-hydroxypyrene. Synchronous Fluorescence Spectroscopy (SFS) was explored as a fast screening method and HPLC was applied as a complementary method for confirmation of the SFS results. In Porcellio scaber the amount of 1-hydroxypyrene exceeded that of pyrene, which indicates that pyrene is being metabolized extensively. SFS and HPLC demonstrated that Porcellio scaber also forms a number of other fluorescent metabolites. These metabolites exhibit fluorescence at the same excitation wavelength as 1-hydroxypyrene, possibly indicating a similar fluorophore structure. Determination of 1-hydroxypyrene is considered as a first step towards an improved environmental risk assessment of PAH exposure, as pyrene is a predominant member of this class of environmental contaminants and 1-hydroxypyrene is its major metabolite.