Abstract Background: The potential for cross-contamination of additives among evacuated blood tubes has led to the development of the order of draw. This practice, however, is mainly based on scarce, anecdotal, and mostly outdated literature data. Therefore, the goal of this investigation was to definitely establish whether or not the indication of a specific order of draw is still justified. Methods: The study population consisted of 57 outpatients referred to the outpatient oral anticoagulant (OA) clinic of the Academic Hospital of Verona and 58 healthy volunteers enrolled from the laboratory personnel. In OA outpatients, one serum tube was collected immediately after needle insertion, followed by a buffered sodium citrate tube and another serum tube. In the healthy volunteers, one serum tube was collected immediately after needle insertion, followed by a potassium-ethylenediaminetetraacetic acid (K₂-EDTA) tube and another serum tube. After separation, the serum was tested for potassium, sodium, calcium, magnesium, and phosphorus in the first and second serum tubes. Results: No significant difference could be observed between the first and the second serum tubes for any of the parameters. The bias calculated with Bland-Altman plots did not achieve statistical significance when the serum tube was collected after either a K₂-EDTA or a sodium citrate tube. Conclusions: According to our data, revision of national and supranational recommendations on blood collection by venipuncture should consider that the order of draw exerts a negligible effect on sample quality, and this aspect should no longer be considered a quality criterion when evaluating the performance of phlebotomists.