In conclusion, we have established a simple, robust, and reproducible method to accurately quantify the concentration and the exact number of molecules that are bound to AuNPs. Deconvolution of UV-visible absorption absorption spectra is highly recommended that can be used in collaboration with DLS, Zeta potential and TEM to build an accurate understanding of the quantity and nature of conjugated molecules on multi-functionalized AuNPs. This method of accurately determining the concentration of each molecule bound to multi-functionalized AuNPs could be used by researchers from various disciplines. For instance, to tailor the material surface and binding concentration based on desired application without misinterpreting the binding concentration.