Cross-sectional biomarker studies can provide a snapshot of the frequency and characteristics of exposure/disease in a population at a particular point in time and, as a result, valuable insights for delineating the multi-step association between exposure and disease occurrence. Three major issues should be considered when designing biomarker studies: selection of appropriate biomarkers, the assay (laboratory validity), and the population validity of the selected biomarkers. Factors related to biomarker selection include biological relevance, specificity, sensitivity, biological half-life, stability, and so on. The assay attributes include limit of detection, reproducibility/reliability, inter-laboratory variation, specificity, time, and cost. Factors related to the population validity include the frequency or prevalence of markers, greater inter-individual variation than intra-individual variation, intra-class correlation coefficients (ICC), association with potential confounders, invasiveness of specimen collection, and subject selection. Three studies are selected to demonstrate different features of cross-sectional biomarker studies: (1) characterizing the determinants of the biomarkers (study I: urinary PAH metabolites and environmental particulate exposure), (2) relationship of multiple biomarkers of exposure and effect (study II: relationship between urinary PAH metabolites and oxidative stress), and (3) evaluating gene-environmental interaction (study III: effect of genetic polymorphisms of GSTM1 on the association of green tea consumption and urinary 1-OHPG levels in shipbuilding workers).