Optimal supply of trace elements (TE) is a prerequisite for microbial growth and activity in anaerobic digestion (AD) bioprocesses. However, the required concentrations and ratios of essential TE for AD biotechnologies strongly depend on prevailing operating conditions as well as feedstock composition. Furthermore, TE in AD bioreactors undergo complex physicochemical reactions and may be present as free ions, complex bound or as precipitates depending on pH, or on the presence of sulfur compounds or organic macromolecules. To overcome TE deficiency, various commercial mineral products are typically applied to AD processes. The addition of heavy metals poses the risk of overdosing operating systems, which may be toxic to microbial consortia and ultimately the environment. Adequate supplementation, therefore, requires appropriate knowledge not only about the composition, but also on the speciation and bioavailability of TE. However, very little is yet fully understood on this specific issue. Evaluations of TE typically only include the measurement of total TE concentrations but do not consider the chemical forms in which TE exist. Thus detailed information on bioavailability and potential toxicity cannot be provided. This review provides an overview of the state of the art in approaches to determine bioavailable TE in anaerobic bioprocesses, including sequential fractionation and speciation techniques. Critical aspects and considerations, including with respect to sampling and analytical procedures, as well as mathematical modeling, are examined. The approaches discussed in this review are based on our experiences and on previously published studies in the context of the COST Action 1302: European Network on Ecological Roles of Trace Metals in Anaerobic Biotechnologies.