We compared various methods of amphibian marking techniques suggested by a University Institutional Animal Care and Use Committee (IACUC) to assess alternatives to toe-clipping without anesthesia. Marking treatments applied to Dusky (Desmognathus fuscus) and Seal (Desmognathus monticola) Salamanders included visible implanted elastomer (VIE) injections, toe-clipping without anesthesia, toe-clipping with a local anesthetic, toe-clipping with whole body anesthesia, and a control group (handled–unmarked). Randomly selected individuals in each group were euthanized and heads and torsos assayed to assess the effect of the marking techniques on stress hormone responses (adrenaline [epinephrine] and noradrenaline [norepinephrine]) levels. Behavioral responses were also observed in a randomly selected group of individuals from each treatment. We found no significant statistical differences in any of the marking methods on the stress hormone responses or on the behaviors of the salamanders. Our findings suggest that toe-clipping (without anesthesia) and VIE do not differ in their respective effects on stress hormone levels linked to the infliction of pain. We suggest that toe-clipping without anesthesia is a viable and humane field technique for marking desmognathine salamanders and similar organisms.