We have prepared negative-to-positive surface charge gradients using a two-step reaction. In the first step a monolayer of 3-mercaptopropyltrimethoxysilane (MTS) was deposited on fused silica and then selectively oxidized by UV exposure to create a surface gradient of sulfhydryl groups. In the second step these sulfhydryl groups were reacted with N-(iodoethyl)-trifluoroacetamide, thus creating a secondary gradient with negative (sulfonate) to positive (amine) surface charges. The negative-to-positive charge gradient surfaces were characterized by contact angle, XPS, and then used to study adsorption of three plasma proteins. Adsorption kinetics of three human plasma proteins, albumin, IgG and fibrinogen, were measured using dual channel total internal reflection fluorescence (TIRF) technique from solutions equivalent to their 1/100 concentrations in plasma. For each protein, two adsorption experiments were carried out in parallel using the same gradient surface. In one TIRF channel the binding of one of three fluorescently labeled proteins was measured from single protein solution, and in the other channel the adsorption of the same protein was measured from the solution mixture with other two unlabeled proteins. The surface adsorption profile for each of the three proteins was determined using autoradiography and applied to quantify TIRF-measured adsorption kinetics and extract the adsorption parameters and the respective binding affinities.