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The Negev Desert tortoise (Testudo werneri) is one of the smallest tortoise species in the Mediterranean region. This is a critically endangered species (CITES I) in its native habitat, which includes the Saharo-Arabian sands of northern Egypt, Sinai, and the Negev Desert in Israel. Great efforts have been invested in captive breeding and reintroduction of this tortoise to the wild. The purpose of this study was to collect blood samples from healthy Negev Desert tortoises kept in well-managed zoologic collections in order to describe hematologic, plasma biochemistry, and acid-base analytes for this species. Data of 36 different blood analytes were collected using the Abaxis Vetscan bench-top analyzer and i-STAT handheld analyzer, and a significant difference was observed between males and females in 13 of the measured analytes. To the authors’ knowledge, this is the first report of blood analytes for the Negev Desert tortoise; however, the data do not fully meet the strict ASVCP guidelines required for reference range determination and thus can only provide a rough estimate for evaluating the health status of Negev Desert tortoises using similar testing methodology.