Detection of proteins in solid-phase assays has been conventionally achieved by fluorescence methods. However, a main disadvantage of fluorescence labeling of proteins is that the introduction of fluorophores may change the proteins physical properties, such charge, hydrophobic/hydrophilic character and structure. To circumvent the problems associated with fluorescent labeling of proteins, several label-free methods have been developed. In this study, we report a label-free spectroscopic method for the detection of proteins utilizing ultraviolet-visible sum-frequency generation (UV-Vis-SFG). The well-established avidin-biotin and anti-biotin antibody (IgG)-biotin systems were used to demonstrate the ability of UV-Vis SFG to detect proteins association to a surface without the need of external labels. In addition, the binding affinities of the avidin-biotin and anti-biotin antibody (IgG)-biotin systems were measured and compared with fluorescence data using fluorescently labeled analogs of the avidin and anti-biotin antibody. The results show that UV-Vis SFG is a valuable technique to directly detect label-free proteins adsorbed at the solid-liquid interface.