Dendrobatids are recognized by the unique diversity of lipophilic alkaloids found on their skin. However, to elucidate the structures of these over 500 alkaloids, more than 10,000 animals of different species have been euthanized in the past 50 years. The gold standard method for chemical profiles of dendrobatids involved the preparation of methanolic extracts of the skin. Therefore, there is an urgent need for non-lethal techniques, which allow the access to chemical profiles of endangered and endemic species. The aim of this study is the accomplishment of two in vivo extraction protocols. First, a swab-based standardization process testing three different materials (rayon, cotton, polyurethane foam) was performed analyzing the chemical profiles from Dendrobates truncatus and Oophaga histrionica by single quadrupole GC-MS. This last species was collected in two localities for comparing variation determined by geographic location. Secondly, we present a comparison of the results obtained from the longitudinal extraction of alkaloids in Dendrobates auratus employing PDMS-patches over six successive collection times analyzed by GC-Q-TOF. Results from skin swabs resulted in similar chemical profiles to those obtained by the gold standard lethal method, detecting a total of 94 compounds. Our findings demonstrate that species and localities could be differentiated based on swab-collection comparisons, despite extraction was not exhaustive. In contrast, semi-quantitative measurements led to underestimation, because many compounds were found at very low intensities. On the other hand, PDMS-patches allowed an in vivo temporal monitoring of 63 compounds. However, after 36 hours of successive sampling of the secretions, the extraction efficiency in terms of abundance and diversity decreased dramatically. These results are especially relevant now, when amphibians are facing panzootic chytridiomycosis and the bio- and chemodiversity of Colombia plays an essential role, being a megadiverse country and having a high number of endemic dendrobatids and/or at risk of extinction.