Direct nanoscale patterning of maleimide-linked biotin on mercaptosilane-functionalized glass substrates using dip-pen nanolithography (DPN) was facilitated by the addition of a small amount of the biocompatible nonionic surfactant Tween-20. A correlation was found between activated biotin transfer from the AFM tip with surfactant included in the ink and an increase in the wettability of the partially hydrophobic silanized substrate. Surfactant concentration represents a new control variable for DPN that complements relative humidity, tip−substrate contact force, scan speed, and temperature. Using surfactants systematically as ink additives may expand the possible ink−substrate combinations that can be used for patterning biotin and other biomolecules, including proteins.