Pseudomonas aeruginosa (P. aeruginosa) causes a broad spectrum of skin infections and early treatment with antibiotics is critical for successful therapy. Currently, P. aeruginosa is detected by time-consuming culturing and counting the bacteria. Therefore, the goal of the present study was to design a rapid, sensitive, and specific protocol for the identification of P. aeruginosa infection in the skin. For this purpose, a methodology was developed which combines isolation of bacteria by species-specific immunomagnetic beads, extraction of DNA by magnetic particles, and confirmation of the identity of P. aeruginosa by real-time PCR. The efficiency of P. aeruginosa extraction from skin samples was greater than 90%, while only minimal amounts of other species were captured by immunomagnetic beads. Extraction of DNA by magnetic particles yielded high-quality DNA which was used in real-time quantitative PCR using an optimized set of primers. The method was highly sensitive as it was capable of detecting as little as 100 cfu of P. aeruginosa. It was also highly specific, as DNA extracted from other species of bacteria did not generate positive signals. Application of this novel rapid methodology to clinical samples yielded results identical to those obtained with traditional methods. Thus, the accumulated results demonstrate unequivocally that a novel rapid, sensitive, and specific method for detection of skin infections with P. aeruginosa was successfully developed and can be utilized clinically.