The purpose of this study was to determine vitamin (A, D, E), mineral (cobalt, copper, molybdenum, manganese, iron, selenium, zinc), and plasma biochemistry concentrations in free-ranging, eastern massasauga rattlesnakes (EMR) (Sistrurus catenatus) in relation to age, sex, reproductive status, and season. Plasma samples were collected from 24 snakes post-brumation (April–May) in Michigan. Significant increases in weight and plasma vitamin E, iron, molybdenum, calcium, phosphorus, triglycerides, and cholesterol were detected in gravid compared to non-gravid females. Vitamin D synthesis from ultraviolet B radiation may be possible in EMR and requires further research. Gravid snakes appear to bioaccumulate molybdenum in tissues. Although the sample size and methodology did not meet ASVCP guidelines for establishing reference ranges, the data provide an important first step in elucidating the role of vitamin and trace minerals in relation to EMR health and reproduction. These data can be used to compare trends across free-ranging and zoo-managed EMR populations and may help to guide changes in husbandry and nutrition programs for the zoo-managed EMR.