To assess the safety of engineered nanomaterials (ENMs) and to evaluate and improve ENMs' targeting ability for medical application, it is necessary to analyze the fate of these materials in biological media. This protocol presents a workflow that allows researchers to determine, characterize and quantify metal-bearing ENMs (M-ENMs) in biological tissues and cells and quantify their dynamic behavior at trace-level concentrations. Sample preparation methods to enable analysis of M-ENMs in a single cell, a cell layer, tissue, organ and physiological media (e.g., blood, gut content, hemolymph) of different (micro)organisms, e.g., bacteria, animals and plants are presented. The samples are then evaluated using fit-for-purpose analytical techniques e.g., single-cell inductively coupled plasma mass spectrometry, single-particle inductively coupled plasma mass spectrometry and synchrotron X-ray absorption fine structure, providing a protocol that allows comprehensive characterization and quantification of M-ENMs in biological matrices. Unlike previous methods, the protocol uses no fluorescent dyes or radiolabels to trace M-ENMs in biota and enables analysis of most M-ENMs at cellular, tissue and organism levels. The protocols can be applied by a wide variety of users depending on the intended purpose of the application, e.g., to correlate toxicity with a specific particle form, or to understand the absorption, distribution and excretion of M-ENMs. The results facilitate an understanding of the biological fate of M-ENMs and their dynamic behavior in biota. Performing the protocol may take 7-30 d, depending on which combination of methods is applied.