Suspicion of catheter-related bloodstream infection (CR-BSI) leads frequently to unnecessary catheter withdrawals, and many catheter-tip cultures yield negative results. The objective of this study was to compare the yield of 3 microbiological procedures to assess CR-BSI without catheter withdrawal. The study was prospectively performed in a group of patients without neutropenia or blood disorders who were admitted to the intensive care unit during a 37-month period with sepsis suspected on clinical grounds and who had central venous catheters inserted for >48 h. The microbiological procedures compared were semiquantitative cultures from hub and skin (superficial), differential quantitative blood cultures, and differential time to positivity between cultures of blood obtained from catheter hubs and peripheral blood. Of the 204 episodes of suspected CR-BSI included in the study, 28 were confirmed to be CR-BSI. We obtained the following results for sensitivity, specificity, positive and negative predictive values, and accuracy: superficial cultures, 78.6%, 92.0%, 61.1%, 96.4%, and 90.2%, respectively; differential quantitative blood cultures, 71.4%, 97.7%, 83.3%, 95.6%, and 94.1%, respectively; and differential time to positivity, 96.4%, 90.3%, 61.4%, 99.4%, and 91.2%, respectively. CR-BSI can be assessed without catheter withdrawal in patients without neutropenia or blood disorders who have catheters inserted for a short time and are hospitalized in the intensive care unit. Convenience, use of resources, and expertise should determine the technique of choice in different medical contexts. Because of ease of performance, low cost, and wide availability, we recommend combining semiquantitative superficial cultures and peripheral vein blood cultures to screen for CR-BSI, leaving differential quantitative blood cultures as a confirmatory and more specific technique.