One challenge facing the laboratory forensic toxicologist today is verifying the validity of the random urine specimen submitted for workplace drugs of abuse analysis. Determining whether urine substitution has occurred is best accomplished through the inspection of the specimen's appearance and the performance of specific laboratory tests, such as determining the concentration of biochemical metabolic waste products and measuring indices of urine concentration. Criteria for classifying submitted urine as substituted are postulated after an extensive review of the published scientific literature. Relevant studies that were evaluated include normal random urine reference interval studies, clinical studies involving the analysis of random urine specimens, theoretical dilutional limits, medical conditions resulting in overhydration, and water-loading studies. After compilation of the study data, derived substituted criteria of urinary creatinine < or = 5.0 mg/dL and urinary specific gravity < or = 1.001 are suggested. A urine specimen meeting these criteria may be considered substituted because it is not consistent with the clinical characteristics associated with normal human urine.