Prosthetic joint infection is a serious complication of total joint arthroplasty that causes great morbidity in affected individuals. The most common cause of prosthesis-associated infections is members of Staphylococcus spp., including Staphylococcus epidermidis. Culture has served as the gold standard for diagnosis, despite obvious shortcomings in terms of sensitivity and time. Bacterial genomic DNA extraction methodologies were evaluated for optimal recovery of genomic DNA from sterilised washout samples, spiked with Staphylococcus epidermidis. Real time PCR assays targeting the Staphylococcus epidermidis specific gseA gene were designed to reliably detect and quantify S. epidermidis. Sixty postoperative washout samples from primary hip and knee arthroplasties were taken aseptically. All were shown to be culture negative using the culture-dependent approach. These samples were subjected to S. epidermidis-specific real time PCR. Standard curve showed good linearity. Sensitivity limit of the assay was less than 10 CFU S. epidermidis/sample. Reproducibility of the assay was confirmed. Staphylococcus epidermidis was not identified in any of the samples taken using the novel species-specific SYBR green real time PCR technique. Results indicated that washout samples were true negatives and did not harbour S. epidermidis. To support this, patients displayed no symptoms of infection. To illustrate the full effectiveness of the novel real time PCR assay, a larger number of samples need to be tested (> 1,000 patients).