A proficiency testing (PT) scheme was prepared for laboratories engaged in bioanalytical testing for synthetic opioid compounds in urine, plasma, and whole blood. Samples were prepared using compounds included in the Opioid Certified Reference Material Kit (Opioid CRM Kit) developed by the U.S. Centers for Disease Control and Prevention. Laboratories received samples during a 2-year project with each year consisting of two PT events 6 months apart. In the first year (pilot test), participants included 10 public health laboratories throughout the United States. In the second year, the group of laboratories expanded to include clinical and forensic drug testing laboratories, and 12 additional participating labs joined the program. In Year 1, overall detection percentages for the compounds present in the PT samples were 95.5% in Event 1% and 97.2% in Event 2. There were 31 apparent false positives reported in Event 1 and four apparent false positives reported in Event 2. Carryover or contamination in laboratory analytical systems were found to be the most significant causes of the false positive results, and none of the laboratories that reported false positives in Event 1 did so in Event 2. In Year 2, overall detection percentages for the compounds present in the PT samples were 89.5% in Event 3% and 94.8% in Event 4. There was one apparent false positive reported in Event 3 and three apparent false positives reported in Event 4. Improvements in drug detection between the two PT events in each year demonstrated the benefit of PT schemes in identifying and addressing potential deficiencies in laboratory systems.