Background: Phlebotomy or drawing of blood sample is one of the initial steps in processing of samples for various investigations of the patients in clinical laboratory. The sample for various investigation has to follow certain protocol or order of blood draw into different vacutainers by phlebotomist or the clinical person drawing the blood to avoid errors in test results. Hence awareness of order of blood draw among them is very essential. Methods: It is a cross sectional and observational study. Based on CLSI H3-A6 (clinical and laboratory standards institute) guidelines, a questionnaire consisting of 13 multiple choice questions was prepared after validation and distributed amongst the nurses, who were on duty during the study. The answers to the questionnaire were analysed using SPSS version 23. Descriptive statistics was done for all the data collected. Results: Total 120 nurses participated in this study in a tertiary care hospital. Nurses who were able to identify Color of the vacutainer with respective to additives (90%), correct order of draw (52%), volume of blood sample collected in vacutainer (62.9%), sample collected directly into vacutainer with vacuum suction (61.7%) was incomplete. Nurses also had wrong practices, where sample was transferred from one vacutainer to other (3.3%), collected the blood sample from the arm which had IV line (28%). Conclusions: In this study, it was found that awareness on the level of order of blood draw among nurses was found unsatisfactory. Frequent training and monitoring of work practices should be developed for nurses to reduce the errors in sample collection.