In order to understand the mechanism of biosensing of field-effect-based biosensors and optimize their performance, the effect of each of its molecular building blocks must be understood. In this work the effect of the self-assembled linker molecules on the top of a biofield-effect transistor was studied in detail. We have combined Kelvin probe force microscopy, current−voltage measurements, and device simulations in order to trace the mechanism of silicon-on-insulator biological field-effect transistors. The measurements were conducted on the widely used linker molecules (3-aminopropyl)trimethoxysilane (APTMS) and (11-aminoundecyl)triethoxysilane (AUTES), which were self-assembled on an ozone-activated silicon oxide surface covering the transistor channel. The work function of the modified silicon oxide decreased by more then 1.5 eV, while the transistor threshold voltage increased by about 30 V following the self-assembly. A detailed analysis indicates that these changes are due to negative-induced charges on the top dielectric layer, and an effective dipole due to the polar monolayer. The results were compared with metal gated transistors fabricated on the same die, and a factor converting the molecular charge to the metal gate voltage was extracted.