An increase in cases of death from overdose and abuse from fentanyl or sufentanil is being encountered by the Bexar County Forensic Science Center in San Antonio, Texas. These drugs have been abused almost solely by health care professionals. The fentanyl derivatives cannot currently be detected by routine laboratory drug-screening programs. Forensic toxicology assays that identify the specific analyte must be used. We report a sensitive assay for detection of fentanyl and sufentanil with a detection limit of -0.5 ng/ml. In addition, results from the analysis of urine and blood samples obtained up to 72 h after drug administration to five patients undergoing cardiac bypass surgery who had received either fentanyl or sufentanil are described. The new procedure enables detection of these drugs more readily, in smaller amounts, and for a longer period of time after use than previously possible. We hope this will lead to intervention and treatment in those abusing the drugs.