The amino acid sequences of some ancestral proteins have been inherited in their descendants with a few modifications. Therefore, in some cases, a phylogenetic tree can be constructed by comparing extant amino acid sequences that were evolved from a single common ancestor. Moreover, ancestral sequences of a particular protein can be inferred using the topology of the phylogenetic tree in combination with the extant amino acid sequences contained in the tree. We recently inferred ancestral amino acid sequences of nucleoside diphosphate kinase that might have existed 3,500–3,800 million years ago. Physicochemical analysis of the experimentally reconstructed ancestral sequences revealed that the resurrected proteins are stable around 100 °C. Given the hyperthermophilicity of ancient organisms, the ancestral sequence reconstruction technique presented in this chapter will serve as a generic method for creating thermally stable proteins.