The objective of this study was to assess the extent of knowledge and application of central line bundles in the intensive care unit (ICU) of a tertiary care hospital for the purpose of avoiding central line-associated bloodstream infections (CLABSI). This assessment was conducted through the use of a questionnaire. A cross-sectional study was conducted in the ICU, involving doctors and nurses. The study was observational in nature. The study employed a methodical validated questionnaire to evaluate the level of knowledge, attitude, and practice of central line bundles for the prevention of central line-associated bloodstream infections (CLABSI). The questionnaire was designed using preexisting awareness surveillance systems, infection control measures, and patient care practices that were specifically relevant to CLABSIs in the ICU. The data were analyzed utilizing SPSS. The research involved a total of 93 healthcare professionals, consisting of 67 physicians and 26 nurses. The mean knowledge score among participants was 82%, with higher scores reported in individuals who had training in central line bundles. Healthcare professionals exhibited robust compliance with hand cleanliness, antiseptic skin preparation prior to insertion, aseptic draping of the patient, utilization of utmost sterile barriers, verification of central venous catheter (CVC) tip placement using chest X-ray or fluoroscopy, and preservation of a sterile environment. The study emphasized the significance of training in enhancing understanding and adherence to central line bundling protocols in ICUs. Participants exhibited a high level of knowledge and commitment to recommended practices, indicating that this training can have a favorable effect on CLABSI rates. Singh S, Sharma A, Dhawan M, Sharma SP. Assessment of the Level of Awareness and Degree of Implementation of Central Line bundles for Prevention of Central Line-associated Blood Stream Infection: A Questionnaire-based Observational Study. Indian J Crit Care Med 2024;28(9):847-853.