We investigated a method for collecting and processing tear samples from loggerhead (Caretta caretta), green (Chelonia mydas), and Kemp's ridley (Lepidochelys kempii) sea turtles and to identify tear biomarkers and potential differences between unaffected sea turtles and those affected by cold stun syndrome. Tear samples from unaffected and cold-stunned loggerhead, green, and Kemp's ridley sea turtles were collected with sterile, cellulose, latex-free ophthalmic eye spears. We pooled spears to achieve acceptable concentrations, which we extracted and analyzed with proton nuclear magnetic resonance spectroscopy. Using principal components analysis, we identified five tear biomarkers (propylene glycol, glycerol, lactate, formate, and an unidentified metabolite) that distinguished unaffected sea turtles from those with cold stun syndrome. The formate concentration was significantly lower (one-sided, exact, two-sample permutation, P=0.019) in unaffected sea turtles, which is consistent with clinical metabolic acidosis reported in cold-stunned animals. Collection of sufficient sample volume for analysis required multiple spears per sample cohort, but tear sample collection from sea turtles was easy to perform and well tolerated by the animals. Sea turtle tears can be an appropriate sample for some metabolomics research questions.