The use of central vascular catheters (CVC) is associated with a substantial number of complications, amongst which infections predominate. A diagnosis of CVC-related infection usually requires catheter removal for culture. Semiquantitative (roll-plate method) and quantitative methods (flush, vortex, centrifugation or sonication methods) are the most reliable diagnostic methodologies requiring catheter removal, because of their greater specificity. The roll-plate method is the simplest and most commonly used technique. This method only samples the external surface of the catheter, and is particularly indicated for recently inserted catheters in which extraluminal colonisation is the primary mechanism of infection. Luminal culture techniques, such as the quantitative methods, may be more relevant for catheters that have been in place for a long period of time. However, in up to 85% of removed CVC the culture is negative, and other diagnostic techniques that do not require catheter removal have been proposed, including paired quantitative blood cultures, endoluminal brushing, and differential time to positivity (DTP) of paired blood cultures. DTP, that compares the time to positivity for qualitative cultures of blood samples simultaneously drawn from the CVC and a peripheral vein, appears to be the most reliable in the routine clinical practice since many hospitals use automatic devices for qualitative blood culture positivity detection. More recently catheter-sparing direct diagnostic methods, which include Gram stain and acridin-orange leucocyte cytospin (AOLC) test, appeared to be especially useful because of the rapidity of results and the ability to distinguish different microorganisms, allowing early targeted antimicrobial therapy.