OBJECT Diagnosis of ventriculostomy-related infections (VRIs) is challenging due to the lack of rapid, sensitive assays for pathogen detection. The authors report the development of a multiplex polymerase chain reaction (PCR) assay for differential diagnosis of common VRI pathogens. METHODS MassTag PCR was used to develop a multiplex assay for detection of 11 VRI pathogens. The assay was established and optimized using cloned template standards and spiked samples and was then evaluated on CSF specimens from ventricular drains. Subjects were grouped into definite VRI, possible VRI, or no VRI based on conventional microbiology, CSF evaluation, and clinical parameters. RESULTS CSF specimens were obtained from 45 subjects (median age 49 years, interquartile range 32–63 years; 51% were male). The assay detected 10–100 genome copies. It detected a pathogen in 100% (6 of 6) of definite VRI cases in which a pathogen targeted by the assay was present; these represented 67% of all definite VRIs (6 of 9). Among subjects with a possible VRI, the assay detected a pathogen in 29% (5 of 17). In subjects without overt infection the presence of a pathogen was detected in 32% of subjects (6 of 19), albeit with lower signal compared with the VRI group. CONCLUSIONS MassTag PCR enabled parallel testing of CSF specimens for 11 pathogens of VRI. The high sensitivity of PCR combined with possible device colonization, specimen contamination, and concurrent antibiotic treatments limit the clinical value of the assay, similar to other current diagnostic approaches. With further optimization, multiplex PCR may provide timely identification of multiple possible VRI pathogens and guide management, complementing classic culture approaches.