Plasma separator tubes (PSTs) are a variant of lithium heparin blood tube containing a polymer gel, which, when centrifuged, creates a physical barrier between plasma and blood cells. Their use is common in laboratory procedures of reptilian species. This study aimed to determine whether the use of plasma separator tubes impacts plasma biochemistry data in green sea turtles (Chelonia mydas) at time of collection and after 24 hr of contact time with the separator gel after centrifugation at refrigerator temperature. A single blood sample was collected from 42 rehabilitating green sea turtles at the Sea Turtle Healing Center, Brevard Zoo, Melbourne, Florida, USA and divided into one lithium heparin tube [LHT (0 hr)] and two PSTs. After immediate centrifugation of all three tubes, plasma was transferred from the LHT (0 hr) and one PST (0 hr) into tubes without additive. The plasma was left in contact with the separator gel in the second PST (24 hr). After 24 hr of refrigeration, all three plasma aliquots were analyzed for the following 23 analytes: sodium, potassium, chloride, carbon dioxide, calcium, phosphorus, magnesium, iron, total protein, albumin, globulin (calculated), aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, lactate dehydrogenase, gamma-glutamyltransferase, creatine kinase, glucose, urea nitrogen, creatinine, uric acid, triglyceride, and cholesterol. No statistically significant differences were found for any biochemical analytes between LHT (0 hr), PST (0 hr), and PST (24 hr). The use of PST does not appear to impact routine plasma biochemical analytes in green sea turtles and analytes appear stable in refrigerated plasma for up to 24 hr after centrifugation when using PSTs.