Background Turtles are a major source of protein for riverside human populations in Brazil. The encouragement of commercial breeding meets conservation efforts for these animals, and it is, therefore, crucial to understand the physiologic and behavioral aspects of semi‐aquatic species in captive conditions. Serum biochemical tests are ancillary diagnostic tools, and sample storage is a main problem since clinical laboratories are not always available near the habitats of these species. Objectives The aim of this study was to provide information about the stability of albumin, aspartate aminotransferase (AST), calcium, creatinine kinase (CK), total cholesterol (Chol), alkaline phosphatase (ALP), gamma glutamyltransferase (GGT), total protein (TP), and urea at different storage times. Methods In all, 17 Arrau turtles (Podocnemis expansa) were used, and the serum obtained was separated into aliquots and analyzed at 0, 4, 8, 16, and 32 days after being stored at −20°C. Results The results showed that albumin, AST, CK, GGT, and TP suffered interference due to the long storage times. Conclusion Analytes such as ALP, calcium, Chol, and urea can be evaluated for up to 1 month after freezing. Albumin, AST, and TP can be analyzed up to 1 week after freezing without alterations, and CK GGT are best evaluated on fresh samples.