For pharmaceutical, biological, and biomedical applications, the functionalization of gold surfaces with pH-sensitive groups has great potential. The aim of this work was to modify gold surfaces with pH-sensitive groups and to determine the pK(a) of the modified gold surfaces using a fluorescent nanoparticle adhesion assay. To introduce pH-sensitive groups onto gold surfaces, we modified gold-coated silicon slides with four different bases: 4-mercaptopyridine (4-MP), 4-pyridylethylmercaptan (4-PEM), 4-aminothiophenol (4-ATP), and 2-mercaptoethylamine (2-MEA). To screen whether the modifications were successful, the binding of negatively charged fluorescently labeled nanoparticles to the positively charged surfaces was visualized by fluorescence microscopy and atomic force microscopy. Next, the pK(a) of the modified surfaces was determined by quantifying the pH-dependent adhesion of the fluorescently labeled nanoparticles with fluorescence spectroscopy. Fluorescence microscopy showed that the gold surfaces were successfully modified with the four different basic molecules. Moreover, fluorescence spectroscopy revealed that fluorescently labeled negatively charged nanoparticles bound onto gold surfaces that were modified with one of the four bases in a pH-dependent manner. By quantifying the adsorption of negatively charged fluorescently labeled nanoparticles onto the functionalized gold surfaces and using the Henderson Hasselbalch equation, the pK(a) of these surfaces was determined to be 3.7 +/- 0.1 (4-MP), 5.0 +/- 0.1 (4-PEM), 5.4 +/- 0.1 (4-ATP), and 7.4 +/- 0.3 (2-MEA). We successfully functionalized gold surfaces with four different basic molecules, yielding modified surfaces with different pK(a) values, as determined with a fluorescent nanoparticle adhesion assay.