NN304 (LysB29-tetradecanoyl, des(B30)-insulin) is a new soluble, long-acting insulin analogue that is tightly bound to human serum albumin differentiating it from human insulin. In the present study, we investigate the effect of fatty acids and selected drugs on the binding of NN304 to human serum albumin in vitro. Binding of the first fatty acid equivalent to albumin does not affect the binding of NN304. None of the tested drugs compete with the binding of NN304 at drug-to-albumin concentration ratios of < 1. The binding of NN304 is shown to be independent of binding of drugs in the two major binding pockets that are located in domains IIA and IIIA of the albumin molecule. Tolbutamide and glibenclamide do not compete with NN304 for binding to albumin at therapeutic drug-to-albumin concentration ratios. High concentrations of acetylsalicylic acid and ibuprofen decrease the affinity of NN304 for albumin, but these interactions occur at drug-to-albumin concentration ratios that are higher than clinically relevant. In conclusion, NN304 is unlikely to be involved in clinically significant drug interactions at the albumin binding level. The unique ligand binding properties of serum albumin and its abundance in the extracellular fluids makes fatty acid acylation and albumin binding an attractive protraction principle for insulin and potentially also for other peptide drugs.