Abstract:Background: Phlebotomy is the system of drawing a blood sample for the use of laboratory testing and for blood transfusion. Professionals who are performing phlebotomy services called phlebotomists. It is a critical part of the pre analytical phase of laboratory testing and is the most neglected procedures in health care. About 70% of the quality of the test are affected during phlebotomy and other pre analytical services. However, little is known about the practice of phlebotomy services in developing countries like Ethiopia. Objectives: To assess the practice of phlebotomists and to identify the major sources of errors during venous blood collection in public hospitals in Addis Ababa. Methodology: Hospital based, cross sectional observational and follow-up study was conducted from March to May 30, 2014. The study followed 40 phlebotomists while each of them was collecting 5 different venous blood collections (giving a total of 200 phlebotomies). Well structured questionnaires and checklists were used to collect data. Data was entered on EPI-Data version 3.1 and statistical analysis was performed with SPSS version 20. Descriptive statistics were employed and the Chi square test was used for comparing major errors observed. Result: Almost all laboratory phlebotomy sites had no Standard Oprating Procedures (SOPs) available in collection sites and most of collection sites were not well ventilated. The major errors identified were use of single glove for more than one client 139 of 200 (69.5%), inappropriate cleaning practice of vein puncture sites 180 of 200 (90%), collecting blood before the disinfectant alcohol dried 139 of 200 (69.5%), incorrect tube collection sequences 107 of 200 (53.5%), unnecessarily applying of tourniquets after blood started flowing in to the collection tubes and syringes 170 of 200 (85.0%) and applying tourniquets before locating and selecting appropriate site for venous blood collection 175 of 200 (87.5%). Conclusion and Recommendation: -Many errors were identified in the phlebotomy practice during the observational study. As the quality of blood specimen influences patient result: emphasis should be given on phlebotomy training to improve the practices for phlebotomists and ensure safety as well as quality during blood collections for laboratory analysis.