Periprosthetic infection is a rare but severe complication of a frequently performed procedure. The diagnosis of this mostly chronic infection is difficult due to the absence of classic signs of infection in one-third of the cases. In this context, periprosthetic infection may be proven by detecting the bacterial pathogen. Aspiration of joint fluid is a suitable method to obtain a representative specimen from the infection site. The puncture must be performed free of contamination, and microbiological processing must respect the special condition of these pathogens. For proof of infection in clinically doubtful cases, cytology of the joint fluid is useful. Aspiration of joint fluid is, apart from biopsy, one of the most important methods for detecting bacterial pathogens in periprosthetic infection. Cultural findings must be interpreted in the context of clinical, histomorphological, cytological, laboratory, and x-ray findings. If doubt remains, the diagnosis should be verified by repeated joint aspiration or by biopsy.