The enzymatic synthesis of the Tn antigen (GalNAc--O-Ser), a glyco-aminoacid of great biological importance, is reported. The reaction was promoted by commercial -N-acetylgalactosaminidase from Acremonium sp., using p-nitrophenyl--N-acetylgalactosamine as the donor. The kinetics were monitored by capillary electrophoresis and LC–UV-MS. For unprotected serine, the role of pH and temperature was investigated, finding that pH 5 and T=18 ◦C gave the best yield. Under these conditions a significant increase of the reaction rate was observed in comparison with previous literature data, using unprotected serine. The role of the bulkiness of the serine protecting groups on the yield was additionally considered, as well as the kinetic profiles generated by the use of two differently protected aminoacids. By proper choice of the protecting group, the reaction yield then increased from 5% (with unprotected serine) to about 50% (with N-Boc and N-methoxycarbonyl serine).