The use of appropriate extraction and amplification controls for acellular specimens is not standardized in the clinical laboratory community. Extraction controls and checks for inhibitors of amplification in cellular specimens are most often accomplished by amplification of an internal human genomic target. This approach is not feasible for acellular specimens, which may contain little or no amplifiable genomic material. Other specimen types, such as stool, frequently contain amplification inhibitors. Failure to test for these inhibitors can result in the reporting of false-negative results. The goal of this study was to evaluate the use of a T4 bacteriophage as an extraction and amplification control for acellular specimens. The T4 bacteriophage assay was evaluated for use as a control in 290 specimens, including cerebrospinal fluid, serum, and filtered stool. Extraction procedures on two automated instruments were assessed, including the Roche MagNAPure Compact (Roche Diagnostics, Indianapolis, IN) and the QIAGEN BioRobot M48 (QIAGEN, Valencia, CA), along with the manual QIAGEN extraction method. The T4 bacteriophage can be extracted reliably and reproducibly from cerebral spinal fluid, serum, and filtered stool and, therefore, is useful as both an extraction control and inhibitor check for these specimen sources.