Emerging contaminants of concern have become a serious issue for the scientific community and society more broadly in recent years due to their increasingly widespread environmental distribution and largely unknown environmental and human health impacts. This study aimed to explore the use of fluorescence excitation-emission (F-EEM) spectroscopy as an alternative analytical method to evaluate the presence of key drugs of addiction (benzoylecgonine, methamphetamine, MDMA, codeine and morphine) in wastewater treatment plants. The chemicals of interest from wastewater were extracted by mixed-mode solid phase extraction and quantified using liquid chromatography tandem mass spectrometry. The same wastewater samples were also analysed by a fluorescence spectrophotometer for fluorescence spectra at wavelengths 280–600 nm (emission) and 200–600 nm (excitation). The study also investigated the relevance of different methods for interpreting F-EEM matrices data including parallel factor analysis (PARAFAC) modelling and fluorescence regional integration technique. PARAFAC identified four components, and among them, component C2, identified at the λex/λem = 275/340 nm wavelength associated with proteinaceous compounds most likely related to tryptophan amino acid, showed significant correlation with codeine removal. MDMA and morphine were not correlated to any of the fluorescence regions. The fluorescence regions related to aromatic protein-like fluorescence were correlated significantly with drug concentration and so may offer a suitable alternative approach for monitoring drugs including benzoylecgonine, methamphetamine and codeine.