Many epidemiological studies have been conducted to determine if any relation exists between pesticide exposure and disease. Biological monitoring is a useful tool for establishing the presence and magnitude of exposures, which are essential parts of the exposure → disease continuum. In the past, we had almost limitless urine for biological measurements, but this has changed dramatically as the study populations continue to focus on young children, for whom urine collection is difficult, and as the number of pesticides for which exposure information is needed has increased. To accommodate the biological monitoring component of these studies, we refined our methods to allow maximum exposure information from a limited-volume urine sample. Using three separate analytical methods, each requiring only 2 mL of urine, we can successfully measure 35 different pesticides or metabolites at background levels with a high degree of selectivity and precision. We describe a comprehensive approach to biological monitoring of current-use pesticides in urine using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and gas chromatography-MS/MS (GC-MS/MS) with quantification using isotope dilution.