We observed unexpectedly high levels of racemization of Fmoc-Ser(tBu)-OH during automated stepwise solid-phase peptide synthesis under standard continuous-flow conditions. We set up a model assay, based on the solid-phase assembly of the tripeptide H-Gly-Ser-Phe-NH2, in order to test the effect of the coupling conditions on serine racemization. Protocols based on collidine as the tertiary base added to the coupling reagent enabled incorporation of serine with less than 1 % racemization.We describe unexpectedly high levels of racemization of Fmoc-Ser(tBu)-OH during automated stepwise solid-phase peptide synthesis under standard continuous-flow conditions. We set up a model assay, based on the solid-phase assembly of the tripeptide H-Gly-Ser-Phe-NH2, in order to test the effect of the coupling conditions on serine racemization. Protocols based on collidine as the tertiary base added to the coupling reagent enabled incorporation of serin with less than 1% racemization.