The ability to accurately determine the number of syringe needle penetration holes through the rubber stoppers in pharmaceutical vials and rubber septa in intravenous (IV) line and bag ports has been a critical factor in a number of forensic cases involving the thefts of controlled substances or suspected homicide by lethal injection. In the early 1990s, the microscopy and microanalysis group of the U.S. Food and Drug Ad-ministration's Forensic Chemistry Center (FCC) developed and implemented a method (unpublished) to locate needle punctures in rubber pharma-ceutical vial stoppers. In 1996, as part of a multiple homicide investigation, the Indiana State Police Laboratory (ISPL) contacted the FCC for in-formation on a method to identify and count syringe needle punctures through rubber stoppers in pharmaceutical vials. In a joint project and investigation using the FCC's needle hole location method and applying a method of puncture site mapping developed by the ISPL, a systematic method was developed to locate, identify, count, and map syringe punctures in rubber bottle stoppers or IV bag ports using microscopic analysis. The method requires documentation of punctures on both sides of the rubber stoppers and microscopic analysis of each suspect puncture site. The final result of an analysis using the method is a detailed diagram of puncture holes on both sides of a questioned stopper and a record of the minimum number of puncture holes through a stopper.