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Aims To explore the relationship between blood sampling techniques and haemolysis. Background Haemolysis rates of blood samples have been thought to be influenced by the method of collection. There is a lack of research evidence available to clearly show the comparative risk of haemolysis across different blood sampling methods, including venepuncture and use of peripheral intravenous cannulas. Design A prospective cohort study. Reporting followed the STROBE checklist. Methods A trained observer was used to record blood sampling techniques over a 10-week period between April and June 2019. These records were then linked to pathology haemolysis results. Multivariable logistic regression was used to model patient and blood draw characteristics affecting haemolysis. Results Most of the blood samples were not haemolysed (n = 324, 87.1%). Multivariable analysis showed haemolysis was associated with increased tourniquet duration and if the level of tube was less than half full. Univariable analysis showed haemolysis was associated with increased age of the patient, the difficulty of cannulation/ venepuncture and increased number of attempts. No difference was found in the haemolysis rate related to the qualification of the blood collector. Conclusion There was no significant difference in haemolysis rates associated with sampling blood from a PIVC compared with venepuncture. Research should be undertaken to determine whether education on the factors influencing haemolysis is useful in decreasing haemolysis rates. Relevance to clinical practice There was no association with increased haemolysis rates when drawing blood via venepuncture compared with a peripheral intravenous cannula. Haemolysis of blood samples was associated with increased tourniquet duration, if level of the tube was less than half-filled, increased age of the patient and difficulty of blood draw. Awareness of the risk of haemolysis associated with specific blood sampling methods may assist clinicians to improve care.