Inking is a chemical defense mechanism which has evolved independently in disparate taxa. This study investigated the L- and D-amino acid composition of ink collected from the in vivo secretions of California sea hares (Aplysia californica), the ink sacs of common cuttlefish (Sepia officinalis) and the colon of pygmy sperm whales (Kogia breviceps). Free amino acids were analyzed by high performance liquid chromatography and fluorometric detection. Ink was prepared for analysis through two methods: traditional filtration through a 0.2 µm nylon filter (VWR) and a new centrifugation method which yielded a higher recovery of amino acids. There was no single or cluster of amino acids which was consistently elevated across the three different inks. The most abundant amino acids were D- and L-alanine in Kogia ink, L-alanine and L-valine in Cuttlefish ink, and L-serine and L-glycine in Aplysia ink. We were able to explain between 1% to 20% of the inks’ dissolved organic carbon (DOC). Due to previous ink composition studies from various taxa, we specifically looked for both free dopamine and L-DOPA but, we did not observe the presence of either compound in any of our ink samples. Instead, we found two unknown fluorescent peaks with similar retention times that may have misidentified as L-Dopa and dopamine in previous studies. The presence of D-amino acids, typically found in the cell walls of bacteria, provides evidence that ink arose within digestive systems or other excretory pathways.