Methyl neo-β-carrabioside has been synthesized for the first time, employing either a late stage ring closure to install the required 3,6-anhydro-bridge or a suitable 3,6-anhydro-galactosyl donor to form the unfavored 1,2-cis-equatorial α-linkage. Using the late stage ring closure approach, an S-linked analogue of methyl neo-β-carrabioside was also realized. These compounds have applications in the identification and characterization of marine bacterial exo-α-3,6-anhydro-d-galactosidases that have specific activity on red algal neo-carrageenan oligosaccharides, such as those found in both family 127 and 129 of the glycoside hydrolases. In addition a biochemical assay using the synthesized methyl neo-β-carrabioside and the marine bacterial exo-α-3,6-anhydro-d-galactosidase ZgGH129 demonstrates that the minimum substrate unit for the enzyme is neo-β-carrabiose.