Microplastics (MPs) are continuously found in soil and water environments. Within aquatic ecosystems, filter-feeding organisms are unable to discriminate MPs from food particles while fish may intentionally ingest MPs by mistaking them for prey. In both cases, MPs can accumulate in tissues with subsequent implications for human and environmental health. The modes of action of MPs are still not fully understood and hence the toxicological effects of these pollutants cannot be fully evaluated. This study aims to improve our understanding of the modes of action and toxicological effects of MPs using a multimodal approach. In the present study, Daphnia magna was deployed as a model to investigate the acute effects of MPs by exposing D. magna specimens for 24 h to fluorophore-coated polyethylene MPs. A multimodal approach, combining fluorescence imaging and mass spectrometry imaging (MSI), was employed to assess the implications of MPs exposures. Fluorescent microscopy revealed a significant accumulation of MPs in the gut of D. magna after acute exposure. Secondary ion mass spectrometry (SIMS) and matrix-assisted laser desorption/ionization (MALDI) imaging were used to study the distribution and potential metabolic effects in exposed D. magna. ToF-SIMS revealed specific fragmentation patterns for polyethylene MPs, with the m/z 43 ion being the most suitable for identifying polyethylene MPs in biological tissue samples. MALDI-MSI showed specific ion types for the eye, gut, optical ganglion, first antennae, and egg tissues of D. magna. MSI data revealed minor alterations in specific regions of D. magna, such as eggs and gut, of D. magna after MPs exposure. The local changes were mainly found in the nucleotide and lipid metabolism within the eggs. In the gut, changes between control and MPs-exposed groups were potentially linked to plastic additives. Overall, the results of this work underline the potential of multimodal approaches based on MSI to study challenging pollutants, such as MPs, and their interactions with tissues.