The field of molecular evolution has benefited greatly from the use of ancestral sequence reconstruction as a methodology to better understand the molecular mechanisms associated with functional divergence. The method of ancestral sequence reconstruction has never been experimentally validated despite the method being exploited to generate high profile publications and gaining wider use in many laboratories. The failure to validate such a method is a consequence of 1) our inability to travel back in time to document evolutionary transitions and 2) the slow pace of natural evolutionary processes that prevent biologists from ‘witnessing’ evolution in action (pace viruses). In this thesis research, we have generated an experimentally known phylogeny of fluorescent proteins in order to benchmark ancestral sequence reconstruction methods. The tips/leaves of the fluorescent protein experimental phylogeny are used to determine the performances of various ASR methods. This is the first example of combining experimental phylogenetics and ancestral sequence reconstruction.