Measuring trace levels of multiple environmental chemicals in biological tissues (i.e., biomonitoring) with a high degree of accuracy and precision is possible thanks to sophisticated analytical chemistry techniques and highly trained laboratory personnel. Selection and validation of biomarkers of exposure are critical. We present examples of the use of biomonitoring in exposure assessment for non-persistent chemicals using phthalates as model compounds. We also discuss several factors relevant to interpreting and understanding biomonitoring data, including the impact of metabolism of the chemicals and matrix composition in the selection of biomarkers of exposure, as well as temporal stability considerations that may affect the biomarkers' concentrations.