Chemical modification of proteins is of growing importance to generate new molecular probes for chemical biology and for the development of new biopharmaceuticals. For example, two approved, long-acting insulin variants are lipidated at the LysB29 side-chain. Acylations of proteins have so far been performed in batch-mode. Here we describe the use of flow chemistry for site-selective acylation of a small protein, insulin. To the best of our knowledge this is the first report on flow chemistry for chemical modification of insulin. The first step was to develop reaction conditions for acylation of Lys B29 that gave a soluble mixture and thus was compatible with flow chemistry in a microreactor; this included selection of a soluble base. Secondly, the conditions, such as reagent ratios and flow rate were optimized. Third, the use of these conditions for the acylation with a wide range of acids was demonstrated. Finally, Boc-protected insulins were synthesized. Insulin remained stable towards these flow chemistry conditions. This use of flow chemistry for the chemical modification of insulin opens the prospect of producing chemically modified biopharmaceuticals by flow chemistry with fewer byproducts.