Micro- and nanoplastic pollution has emerged as a global environmental problem. Moreover, plastic particles are of increasing concern for human health. However, the detection of so-called nanoplastics in relevant biological compartments remains a challenge. Here we show that Raman confocal spectroscopy-microscopy can be deployed for the non-invasive detection of amine-functionalized and carboxy-functionalized polystyrene (PS) nanoparticles (NPs) in Daphnia magna. The presence of PS NPs in the gastrointestinal (GI) tract of D. magna was confirmed by using transmission electron microscopy. Furthermore, we investigated the ability of NH2-PS NPs and COOH-PS NPs to disrupt the epithelial barrier of the GI tract using the human colon adenocarcinoma cell line HT-29. To this end, the cells were differentiated for 21 days and then exposed to PS NPs followed by cytotoxicity assessment and transepithelial electrical resistance measurements. A minor disruption of barrier integrity was noted for COOH-PS NPs, but not for the NH2-PS NPs, while no overt cytotoxicity was observed for both NPs. This study provides evidence of the feasibility of applying label-free approaches, i.e., confocal Raman mapping, to study PS NPs in a biological system.