BackgroundCholinesterase is a biomarker for poisonings by anticholinesterase agents, but its reference values are scarce, and possible interaction with collars containing parasiticides has not been studied.ObjectivesWe aimed to evaluate the serum cholinesterase activity of healthy dogs without a history of contact with anticholinesterase agents and healthy animals exposed to commercial collars containing organophosphate.MethodsNinety‐nine dogs were used and included healthy animals without recent exposure to anticholinesterase agents and healthy animals previously exposed to diazinon collars. Serum quantification of the enzyme butyrylcholinesterase (BuchE) through spectrophotometry was conducted on all samples. In experiment 1, BuchE activity was quantified at time 0 and 7 days after, a time when the samples were kept at −18°C. In experiment 2, sampling times were 0, 7, 14, 21, 28, and 56 days.ResultsTime 0 values were 4622.38 ± 1311.53 U/L. After 7 days, a significant decay was observed, with a mean of 3934.45 ± 1430.45 U/L. Spearman's test was performed, finding a weak correlation between ALT, creatinine, total plasma proteins, age, weight, red blood cells, platelets, leukocytes, and BuchE activities. In experiment 2, the mean at time 0 was 4753 ± 454.8 U/L. With exposure to the collar, there was a decay of up to 93% after 14 days.ConclusionsNormality values of serum BuchE in healthy dogs without a history of exposure to anticholinesterase agents were 4360.8–4883.96 U/L. Freezing serum caused a decrease in BuchE activity. Exposure to commercial collars containing diazinon also reduced BuchE activity without clinical signs, indicating that previously exposed animals should be evaluated carefully.