Nanomaterials are materials in which at least one of the three dimensions ranges from 1 to 100 nm, according to the International Organization for Standardization (ISO). Nanomaterials can be categorized according to various parameters, such as their source, their shape, and their origin. Their increasing use in industrial settings, everyday items, electronic devices, etc. poses an environmental and biological risk that needs to be assessed and appropriately addressed. The development of reliable analytical methods for both characterization and quantification of nanomaterials in various matrices is essential. This review summarized the recent trends in analytical methodologies for the characterization and determination of nanoparticles in biological matrices.