The measurement of serum total protein (TP) is commonly performed by the biuret method. Refractometery which is less expensive and faster method also determines serum protein; however its accuracy is controversial. Therefore the primary objective of this study was to investigate portable refractometer as an alternative method to the biuret assay for measuring total serum proteins in horse serum. Serum from 50 male horses admitted to the society of the protection of animals’ abroad (SPANA) clinic was collected and measurement of serum total protein determined using portable refractometer and biuret method. The statistical differences between both methods were analyzed using a paired t-test and the correlation was determined by spearman correlation test. The results of this study demonstrate that there were no statistically significant variations in the mean total protein measured with the refractometer and biuret method (P > 0.05). The correlation between two methods using spearman correlations revealed high correlation between refractometry and biuret assay measurements of TP concentration r = 0.855. Bland–Altman plots indicated that biases indicating the analytic error was (4/50) 8% which is less than allowable total error for total protein determination (< 10%). Therefore portable refractometery can be used as an alternative to biuret method for serum total protein in horse as it is easy to use, quick turnaround time, and is inexpensive.