Globally, industrial discharges with a high xenobiotic load are discharged to water sources. For example, in the Colombian Orinoco region, at the wellhead ~ 16 barrels of water are produced for each barrel of oil. Even after treatment, the discharged waters contain substantial amounts of Polycyclic Aromatic Hydrocarbons (PAHs). These compounds are highly toxic and of global environmental priority, due to their persistence in the environment and accumulation in the food chain. In this work, an analytical excitation-emission fluorescence (EEMS) technique was developed for the detection and quantification of HAPs, and their metabolites in a complex biological matrix (fish bile). This spectroscopic technique is sensitive, faster and less expensive than chromatography. The EEMS allowed detecting the presence of metabolites of PAHs (1-hydroxypyrene, 1- and 2-hydroxynaphthalene, 9-hydroxyphenanthrene) in fish exposed in the laboratory (Piaractus brachypomus and Aequidens metae). High spectral specificity (fluorometric fingerprint) and high sensitivity (up to ppt) were found for the measured compounds and mixtures (precursors, metabolites and bile). For this, a metrological optimization of the fluorometer was made and of the measurement methodology (including the solvent). A high species-species and individual-individual variability was found in the spectral surfaces of the bile. Despite this, it was possible to detect and spectrally quantify the presence of metabolites in bile from their reference spectra (pure compounds in ethanol), with excellent precision (r2> 0.9). The internal filter effects were appropriately captured by a mathematical model.