In environmental epidemiology, use of biomonitoring (i.e., trace-level measurement of environmental chemicals or their metabolites in biospecimens) for exposure assessment has increased considerably in past decades. Although exposure biomarkers should reflect a person’s exposure to the target chemicals (or their precursors) within a specific timeframe, timing, duration, and intensity of exposures are normally unknown and likely vary within the study period. Therefore, evaluating exposure beyond a single time point may require collecting more than one biospecimen. Of note, collection and sample processing procedures will impact integrity and usefulness of biospecimens. All of the above factors are fundamental to properly interpret biomonitoring data. We will discuss the relevance of the exposure assessment study protocol design to (a) ensure that biomonitoring specimens reflect the intended exposure, (b) consider the temporal variability of concentrations of the target biomarkers, and (c) facilitate the evaluation of accuracy and comparability of biomonitoring results among studies.