ABSTRACT Amphibian skin secretions are a rich source of antimicrobial peptides that have long interested both the biomedical and herpetological research communities. While anuran skin peptides are well studied, little is known about skin peptides in salamanders, in part because methods for collecting them are poorly developed. In anurans, pharmacological agents are commonly used to stimulate skin peptide responses, thus increasing the amount of peptide collected. It was unclear whether similar methods would work for salamanders. We conducted a series of experiments to test the effects of immersion in pharmaceutical agents (norepinephrine hydrochloride [NE]; acetylcholine chloride [Ach]), handling, and the interaction of both on peptide collection from the skin of Red-Spotted Newts ( Notophthalmus viridescens ) and Allegheny Mountain Dusky Salamanders ( Desmognathus ochrophaeus ). Our results suggest that vigorous handling, but not immersion in NE or Ach, is a better method for collecting salamander skin peptides. Despite the widespread use of pharmaceuticals to collect anuran skin peptides, we found little evidence that NE or Ach induces peptide release in salamanders. In fact, we show that inclusion of NE in collection solutions interferes with peptide quantification in protein assays.