The main purpose of the present work is to provide a fully integrated temperature control of in-line tryptic digestion in order to facilitate the quality control of polypeptidic therapeutic compounds. The in-line enzymatic reaction was performed in 100 mM bicarbonate ammonium whereas a mixture of citric acid/epsilon-amino caproic acid (pH 5.0 and I 75 mM) was used as a background electrolyte (BGE). After the injection of all reactants (substrate, enzyme, proteolysis buffer), a BGE plug was injected to push all reactants until a position where the capillary is thermostated. Then, the enzymatic reaction was initiated during 15 min of incubation time and finally, a voltage was applied to separate the generated proteolysis products. The methodology was developped regarding the effects of the BGE plug length and pressure on the reactants plug mixing and on the advanced of the tryptic digestion. Successful temperature control of in-line proteolysis with excellent repeatability was obtained in optimal cleavage and separation conditions.