Red Sea sponges offer a potential for production of novel drugs and prototypes. The genus Negombata is a type of sponges abundant in the Red Sea. This sponge produces latrunculins that have well documented antitumor activity in addition to antimicrobial and antiviral effects. The identification of Negombata species is based on morphology and microscopical examination of megascleres of spicules. However, these criteria have proven to be unreliable for identification. Therefore, this study was established to test the accuracy of the spicules based taxonomy against molecular and protein profiles for the two species of Negombata: N. magnifica and N. corticata. About 700 bp of cytochrome c oxidase I gene was sequenced from the tissues of the two Negombata species. Additionally total proteins were extracted from Negombata samples collected from different locations during different seasons and separated by denaturing polyacrylamide gel electrophoresis. Characteristic different protein profiles were obtained for both species. The data obtained from cytochrome c oxidase I gene sequencing and protein profiles can reliably differentiate between different species of Negombata in the Red Sea.