The biochemical parameters of laboratory animals may vary between different lineages and strains of a given species, being influenced by the microenvironment and macroenvironment of the vivarium for breeding and maintenance, as well as particularities of the method and commercial kit used in the analysis. This study aimed to determine the reference values for the hepatic and renal parameters of Unib-line Wistar rats. Forty healthy male Wistar rats were studied, weighing an average of 200 to 300g, from the Laboratory Animal Center of Federal Fluminense University. Quantitative biochemical analysis of the liver profile (AST, ALT, alkaline phosphatase, GT gamma, total proteins, albumin, total bilirubin, and direct bilirubin) and renal markers (urea and creatinine) was performed. The research data established liver and kidney analyte values that were compared with those described in the literature. From the standardization of reference values, it will be possible to optimize the time of experiments with Wistar rats as well as to reduce the number of animals used in the research.