The objective of human biomonitoring is to assess the population’s exposure to organic and inorganic chemicals in order to establish reference values, compare exposure levels among subpopulations, identify temporal or geographic trends, or evaluate the impact of management measures. To this end, the measurement of these biological markers of exposure (i.e., biomarkers) must be based on harmonized methodologies to minimize analytical bias and ensure comparability of results. This requires, in particular, determining the chemical form in which the biomarker should be analyzed (parent compound or metabolites) and using the analytical method most appropriate for the selected biomarker. Results must be standardized to minimize intra- and inter-individual physiological variations, thereby enabling better interpretation and comparability. In this article, we address and discuss issues related to the analytical challenges mentioned above. First, we discuss how taking into account the toxicokinetics of the substances in question can help select, depending on the biological matrix under consideration, the most relevant biomarker (parent compound or metabolites) to analyze in terms of detection frequency, concentration, and specificity, or even cyclic urinary excretion profiles. We then address the issue of analytical methods and discuss how various parameters—such as sample storage, the choice of a deconjugation method, sample preparation, or the analytical workflow—can influence analytical results. Finally, we discuss how taking into account physiological parameters specific to individuals in the study population, for biological matrices such as blood, urine, and hair, can significantly affect the calculation of the concentration of the xenobiotic in question.