The presence of pharmaceuticals and personal care products (PPCPs) in natural water resources due to incomplete removal in Wastewater Treatment Plants (WWTPs) is a serious environmental concern at present. In this work, the effects of three pharmaceuticals (propranolol, triclosan, and nimesulide) on Gammarus pulex metabolic profiles at different doses and times of exposure have been investigated by liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS). The complex data sets generated in the different exposure experiments were analyzed with the ROIMCR procedure, based on the selection of the MS regions of interest (ROI) data and on their analysis by the Multivariate Curve-Resolution Alternating Least Squares (MCR-ALS) chemometrics method. This approach, allowed the resolution and identification of the metabolites present in the analyzed samples, as well as the estimation of their concentration changes due to the exposure experiments. ANOVA Simultaneous Component Analysis (ASCA) and Partial Least Squares Discriminant Analysis (PLS-DA) were then conducted to assess the changes in the concentration of the metabolites for the three pharmaceuticals at the different conditions of exposure. The three tested pharmaceuticals changed the concentrations of metabolites, which were related to different KEGG functional classes. These changes summarize the biochemical response of Gammarus pulex to the exposure by the three investigated pharmaceuticals. Possible pathway alterations related to protein synthesis and oxidative stress were observed in the concentration of identified metabolites.