Pesticides are widely used chemicals making human exposure to pesticides a realistic possibility. Biomonitoring is a common and useful tool for assessing human exposure to pesticides. For the past several decades, methods have been developed to measure a variety of pesticides in human urine or blood to evaluate exposures. Many of these methods have employed analytical techniques such as chromatography and mass spectrometry to accurately measure urinary metabolites or blood body burdens of several classes of pesticides. The pesticides assessed include the banned organochlorine pesticides, the more modern organophosphorus, carbamate, and pyrethroid insecticides along with a variety of herbicides including phenoxyacetic acids and triazines. These methods are capable of detecting concentrations in biological samples resulting from occupational exposures to pesticides, and in some instances, general background exposures from residential or dietary exposures. These data have been used for a variety of applications. They have documented the pervasiveness of pesticide exposures, have allowed us to determine the primary predictors of exposure in certain populations, have helped us to identify the most important pathways of exposure, and have helped us to better understand any potential risks associated with exposures. In addition, these methods have helped us to document poisoning cases and identify etiologic agents in crisis situations. We discuss the methods that have been employed over the last 40 years and how these methods have addressed critical public health questions.