Particle-based assays are becoming versatile analytical tools due to their cost-effectiveness, speed, straightforward and diverse functionalization chemistries, especially when polystyrene particles are used. The introduction of functional groups (-COOH, -NH2, etc.) to the surface of such polystyrene particles promotes their application in bioanalytics. However, the traditional method to determine the amount of surface carboxylate groups is conductivity titration, which is usually time- and resources-consuming and discontinuous. Here, we synthesized polystyrene microparticles with different contents of carboxylate groups, and then investigated a simpler and potentially continuous approach to determine the amount of surface carboxylate groups by Zeta potential measurements. The results were compared to the traditional titration method and to actual coupling efficiencies of the functionalized particles with a model oligonucleotide probe as determined by flow cytometry. All quantification methods revealed good agreement.