Hypotheses about the functions of ancient proteins and the effects of historical mutations on them are often tested using ancestral protein reconstruction (APR)-phylogenetic inference of ancestral sequences followed by synthesis and experimental characterization. Usually， some sequence sites are ambiguously reconstructed， with two or more statistically plausible states. The extent to which the inferred functions and mutational effects are robust to uncertainty about the ancestral sequence has not been studied systematically. To address this issue， we reconstructed ancestral proteins in three domain families that have different functions， architectures， and degrees of uncertainty; we then experimentally characterized the functional robustness of these proteins when uncertainty was incorporated using several approaches， including sampling amino acid states from the posterior distribution at each site and incorporating the alternative amino acid state at every ambiguous site in the sequence into a single "worst plausible case" protein. In every case， qualitative conclusions about the ancestral proteins' functions and the effects of key historical mutations were robust to sequence uncertainty， with similar functions observed even when scores of alternate amino acids were incorporated. There was some variation in quantitative descriptors of function among plausible sequences， suggesting that experimentally characterizing robustness is particularly important when quantitative estimates of ancient biochemical parameters are desired. The worst plausible case method appears to provide an efficient strategy for characterizing the functional robustness of ancestral proteins to large amounts of sequence uncertainty. Sampling from the posterior distribution sometimes produced artifactually nonfunctional proteins for sequences reconstructed with substantial ambiguity.