The C57BL/6J mouse strain is widely used as a common genomic background for many gene-modified murine models. However, few data on its clinical biochemistry are available. Therefore, we conducted a study to provide new protocols for serum biochemical screening and developed the reference range for a set of 13 analytes that pertain to lipoprotein metabolism, electrolyte balance, and data reflecting function of the heart, liver, kidneys, and pancreas. Male and female mice were studied, and blood samples were obtained at six and 20 weeks of age. Of 13 parameters studied, 12 were affected by age and sex. Briefly, male mice had higher triglycerides, cholesterol, high-density lipoprotein cholesterol, glucose, and amylase values. With age, mice of both sexes developed higher triglycerides and glucose concentrations, as well as aspartate and alanine transaminase activities. A significant difference between mice and humans was noted for amylase activity, which is extremely high in this healthy mouse strain. Therefore, we suggest that caution should be taken when data are interpreted to indicate gastrointestinal disease in murine models. The reference values for murine clinical biochemical analytes obtained during the study reported here should be useful for characterizing the biochemical phenotype of experimental mice.