Abstract Objective: Ammonia is extremely unstable in blood specimens and has special requirements during transport, processing and storage. The aim of our study was to determine the stability of ammonia in EDTA K3 blood samples and to establish a protocol for sample handling. Methods: In this study, 36 healthy subjects and 47 inpatients diagnosed with type 2 diabetes mellitus were enrolled. Two peripheral blood samples were collected from healthy volunteers (Sample A1 and A2) and one peripheral blood sample was collected from the inpatients diagnosed with type 2 diabetes mellitus (Sample B). Sample A1 and B were transported in ice bath within 15 minutes of blood collection, centrifuged immediately and processed. The sample was re-centrifuged after 15 minutes and a second ammonia result was obtained. Sample A2 was transported at room temperature and stored between 2 and 4 hours, centrifuged and plasma ammonia measurement was performed. The sample was re-spun after 15 minutes and a fourth ammonia result was obtained. Results: In our study, in healthy group the difference between sample A2 and set point value (on ice, 15 minutes) is 25.08 µg/dl, showing an increase of 55.29%. After another 15 minutes, an increase of 82.02% was observed compared with the standard value. In diabetes mellitus group, after 30 minutes of blood collection, an increase of 11% over the set point value was observed. Conclusions: The blood specimen should be transported on ice to the laboratory and analyzed within 15 minutes of blood collection due to plasma ammonia spontaneously increase.