In veterinary medicine, protein electrophoresis has become a routine test. However, its use in exotic animals remains controversial because of a lack of reference values for many species. Establishing protein electrophoresis reference values of healthy Hermann's tortoises (Testudo hermanni) could prove to be a very useful tool for assessing the health status of this endangered species. The aim of this study was to establish a preliminary reference interval for the protein fractions of Hermann's tortoises under normal conditions. Serum samples from healthy adult Hermann's tortoises (n = 22) were analyzed by cellulose acetate electrophoresis. Total protein concentration (min–max: 2.10–6.20 g/dl) was measured by the biuret method. Four protein fractions were identified: albumin, α-globulins, β-globulins, and γ-globulins. The reference intervals for healthy tortoises were determined by parametric methods as recommended by the American Society of Veterinary Clinical Pathologists Quality Assurance and Laboratory Standards Committee. Furthermore, differences between sexes (15 females, 7 males) were evaluated and a statistical difference was noted. To our knowledge, this is the first study to determine reference intervals for the protein profile of Hermann's tortoise using parametric methods.