Extracts of amphibian skin have been used for centuries in folk medicine and witchcraft due to their possession of a wide spectrum of pharmacological effects. Amphibian skin is a morphologically and physiologically complex organ that plays very important roles in their survival and act as the first line of defence against physical injury and fatal invasion by microorganisms or parasites and against predators. The amphibian skin secretions are complex, species specific cocktails of biological active molecules. Today, the skin secretion is known as a highly-complex mixture of abundant and diverse bioactive molecules and its study has attracted increasing attention over recent years. In this thesis, the skin secretion of the orange-legged leaf frog (Phyllomedusa hypochondrialis) and the Mexican leaf frog (Pachymedusa dacnicolor) were studied by isolating the skin secretion mRNA and establishing a cDNA library. On one hand, a “shotgun” cloning procedure was performed and the resultant peptide precursor-encoding cDNAs that were cloned. On the other hand, the mature active peptides were confirmed by using RP-HPLC, MALDI-TOF mass spectrometry and LCQ-Fleet electrospray ion-trap mass spectrometry. The confirmed mature peptides were then chemically-synthesised and were used to perform a range of biological assays, including antimicrobial assays, haemolysis assays, smooth muscle activity assays and anticancer assays.