Microplastic (MP) pollution is a persisting global problem. Accurate analysis is essential in quantifying the effects of microplastic pollution and develop novel technologies that reliably and reproducibly measure microplastic content in various samples. The most common methods for this are FTIR and Raman spectroscopy. Coloured, standardized beads are often used for method validation tests, which limits the conclusions to a very specific case rarely observed in the natural environment. This study focuses on the preparation of reference micro- and nanoplastics via cryogenic milling and shows their use for FTIR and Raman method validation studies. MPs can now be reproducibly milled from various plastics, offering the advantages of a better representation of MPs in real environment. Moreover, this study highlights issues with the current detection methods, up to now considered as the most reliable ones for MP detection and identification. Such issues, e.g. misidentification, will need to be addressed in the future. Additionally, milled MPs were used in experiments with commercial high-resolution imaging device, enabling a possible in-situ optical detection of microplastics. These experiments represent a step forward in understanding MPs in a water sample and provide a basis for a more accurate detection and identification directly from water, which would considerably reduce the time of analysis.