Objectives: Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has considerable applicative potential for both qualitative and quantitative analyses of elemental spatial distribution and concentration. It provides high resolutions at pg-level detection limits. These qualities make it very useful for analyzing biological samples. The present study responds to the growing demand for adequate analytical methods which would allow to assess the distribution of nanostructured molybdenum(IV) disulfide (MoS₂) in organs. It was also motivated by an apparent lack of literature on the biological effects of MoS₂ in living organisms. The study was aimed at using LA-ICP-MS for comparing micro- and nanosized MoS₂ ditribution in selected rat tissue samples (lung, liver, brain and spleen tissues) after the intratracheal instillation (7 administrations) of MoS₂ nano- and microparticles vs. controls. Material and Methods: The experimental study, approved by the Ethics Committee for Animal Experiments was performed using albino Wistar rats. This was performed at 2-week intervals at a dose of 5 mg/kg b.w., followed by an analysis after 90 days of exposure. The MoS₂ levels in control tissues were determined with the laser ablation system at optimized operating conditions. The parameter optimization process for the LA system was conducted using The National Institute of Standards and Technology (NIST) glass standard reference materials. Results: Instrument parameters were optimized. The study found that molybdenum (Mo) levels in the lungs of microparticle-exposed rats were higher compared to nanoparticle-exposed rats. The opposite results were found for liver and spleen tissues. Brain Mo concentrations were below the detection limit. Conclusions: The LA-ICP-MS technique may be used as an important tool for visualizing the distribution of Mo on the surface of soft samples through quantitative and qualitative elemental mapping. Int J Occup Med Environ Health. 2024;37(1):18–33