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This study aimed to investigate the differences in assessment of total proteins between the use of refractometric and biuretic methods in equine serum and plasma samples and to test the validity of the two methods at different storage conditions. Serum and plasma total proteins concentration from 60 nonhemolytic and nonlipemic equine blood samples was assessed using refractometric and biuretic methods. On serum and plasma samples albumin, triglycerides, cholesterol, glucose, total bilirubin, and fibrinogen values were also evaluated. Samples were analyzed immediately after centrifugation (T0), refrigerated at 4°C and analyzed after 24 hours (T1) and 48 hours (T2), stored under refrigeration at 4°C, and after 1 week (T3) of storage at −20°C. No statistically significant effect of method (P > .05) on serum and plasma total proteins levels was found. A significant effect of storage condition (P < .01) and sample type (P < .05) was revealed on total proteins values. A significant effect of storage condition (P < .01) was found on albumin, triglycerides, glucose, and total bilirubin values measured in both serum and plasma samples. Significant lower fibrinogen values were found at T0 with respect to T1, T2, and T3 and at T1 and T2 with respect to T3 (P < .001). The use of refractometer for total proteins measurement in equine serum or plasma samples without any visible abnormality was sufficiently reliable, providing an inexpensive and rapid alternative to biuret method. The present study suggests that storage conditions of serum/plasma samples are important preanalytical variables, which may considerably affect the determination of the most frequently texted parameters in the equine species.Equine total proteins evaluation through refractometer and biuret methods.Storage conditions and sample type influenced total proteins levels.The methods no significantly influenced the serum and plasma total proteins levels.Refractometric method for total proteins in horse serum/plasma is suitably reliable.