The reactions between 2,3‐epoxypropan‐1‐ol (glycidol) and either the nonglycosylated protein bovine serum albumin (BSA) or the glycoprotein avidin (AV) allowed the successful introduction of diol groups through the addition of some of the proteins' nucleophilic residues to the epoxide ring. The extent of glycolation could be readily tuned by varying the reaction time, the pH of reaction and the concentration of glycidol. Treatment of the stable protein‐diol intermediates with sodium periodate resulted in the generation of reactive aldehyde functionalities through the oxidation of the glycol moieties. At this step, the amount of generated aldehydes could also be modulated by changing the time and/or pH of oxidation. As an application, both formylated proteins were successfully labelled with the hydrazide‐containing fluorescent dye Lucifer Yellow CH (LyCH), affording highly fluorescent conjugates. Mild oxidation of the avidin‐diol left untouched glycol moieties that contributed to greatly enhance water solubility in the avidin‐LyCH bioconjugates. In certain cases the avidin‐LyCH conjugates retained a good affinity for biotin, as shown in a competitive binding assay. Glycidol thus appears to be an attractive low‐cost reagent for protein chemical activation aimed at further coupling of amine‐containing species.(© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)