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The potential role of serotonin (5-HT) as a marker for identifying sensitivity to clinical diseases or behavioral disorders and for monitoring treatment emphasizes the need for reference values of serum 5-HT in dogs. Preanalytical factors such as patient variables, time of sample collection, specimen handling, storage time, and temperature can potentially cause variations to laboratory results. The aim of the present study was to evaluate canine serum 5-HT concentration measured by enzyme-linked immunosorbent assay and to propose standardized preanalytical conditions. Blood samples were collected from the cephalic vein, between 9.00 AM and 11.00 AM, from 120 healthy fasted dogs; the samples were then stored into anticoagulant-free tubes and centrifuged. The serum was stocked at −20°C and analyzed by enzyme-linked immunosorbent assay within 3 months from collection. Because the results were not normally distributed in the reference population, the 2.5 and 97.5 percentiles were calculated to characterize the 95% reference intervals (201-650 ng/mL, 361 median). For statistical analysis, the results were organized into 3 different groups based on the age of the dogs: 1-2 years old (n = 35), 3-7 years (n = 43), and 8-12 years (n = 42). 5-HT levels were higher in 3-7 years group than in other age groups, but no significant age-related dissimilarities were found (Kruskal-Wallis test, P = 0.08). The results of the present study contributed to establish reference intervals for serum 5-HT in dogs which could support both clinical researchers and veterinary behaviorists in designing studies and monitoring the effects of medications. Furthermore, we provide recommendations to decrease 5-HT measurement variation depending on preanalytical factors, to improve reliability and validity of serum measurements. [ABSTRACT FROM AUTHOR]