Background: Despite the availability of established guidelines for measuring platelet serotonin, these guidelines may be difficult to follow in a hospital setting where time to processing may vary from sample to sample. Purpose: The purpose of this study was to evaluate the effect of the time to processing of human blood samples on the stability of the enzyme-linked immunosorbent assay (ELISA) for the determination of platelet serotonin levels in human plasma. Method: Human blood samples collected from a convenience sample of eight healthy volunteers were analyzed to determine platelet serotonin levels from plasma collected in ethylene diamine tetra acetic acid (EDTA) tubes and stored at 4°C for 3 hr, 5 hr, 8 hr, and 12 hr. Results: Refrigeration storage at 4°C for 3 hr, 5 hr, 8 hr, and 12 hr altered the platelet serotonin measurement when compared to immediate processing. The bias for the samples stored at 4°C for 3 hr was 102.3 (±217.39 ng/109 platelets), for 5 hr was 200.1 (±132.76 ng/109 platelets), for 8 hr was 146.9 (±221.41 ng/109 platelets), and for 12 hr was −67.6 (±349.60 ng/109 platelets). Discussion: Results from this study show that accurate measurement of platelet serotonin levels is dependent on time to processing. Researchers should therefore follow a standardized laboratory guideline for obtaining immediate platelet serotonin levels after blood sample collection.