Background: Bufonid parotoid macrogland secretion contains several low molecular mass molecules, such as alkaloids and steroids. Nevertheless, its protein content is poorly understood. Herein, we applied a sample preparation methodology that allows the analysis of viscous matrices in order to examine its proteins. Methods: Duttaphrynus melanostictus parotoid macrogland secretion was submitted to ion-exchange batch sample preparation, yielding two fractions: salt-displaced fraction and acid-displaced fraction. Each sample was then fractionated by anionic-exchange chromatography, followed by in-solution proteomic analysis. Results: Forty-two proteins could be identified, such as acyl-CoA-binding protein, alcohol dehydrogenase, calmodulin, galectin and histone. Moreover, de novo analyses yielded 153 peptides, whereas BLAST analyses corroborated some of the proteomicidentified proteins. Furthermore, the de novo peptide analyses indicate the presence of proteins related to apoptosis, cellular structure, catalysis and transport processes. Conclusions: Proper sample preparation allowed the proteomic and de novo identification of different proteins in the D. melanostictus parotoid macrogland secretion. These results may increase the knowledge about the universe of molecules that compose amphibian skin secretion, as well as to understand their biological/physiological role in the granular gland.Coordena????o de Aperfei??oamento de Pessoal de N??vel Superior (CAPES)Conselho Nacional de Desenvolvimento Cient??fico e Tecnol??gico (CNPq)Funda????o de Amparo ?? Pesquisa do Estado de S??o Paulo (FAPESP)CAPES: 969130CNPq: 406385/2018-1; 303792/2016-7; 440954/2017-7FAPESP: 13/07467-1; 13/07467-1