The ability to functionalize AuNPs with molecules has led to an interest in designing more complex systems with multiple molecule types conjugated to each AuNP, known as multifunctionalized AuNPs. These multi-functionalized AuNPs have a significant advantage over AuNPs conjugated with just one type of molecule as they can be used for more broad applications. An example of multi-functionalized AuNPs is in drug delivery systems, where a targeting agent and drug are conjugated to the surface of the particle to direct the effect of the drug to a specific cell type or region. Another example would be in cancer treatments where multi-functionalized AuNPs can deliver two anticancer drugs to the same targeted tissue simultaneously. The concentration of conjugated molecules on the surface of the AuNPs is an important factor that determines its nano-bio interactions and eventually its biological function. In pharmaceutical applications, the concentration of the conjugated molecules is a significant factor in modulating drug dose thresholds. In biosensing, the minimum concentration of analyte that can be detected is known as its limit of detection or sensitivity. Accurately knowing the working concentrations and how sensitive a biosensor is essential to its accurate function. Consequently, there is a need to develop a simple, robust, and fast analytical method to facilitate surface quantification of AuNPs functionalized with more than one molecule. We have recently reported on the development of a multi-functional AuNP system functionalized with Cyt C for cell-induced apoptosis. Cyt C is a biomolecule of interest due to its redox properties that makes it useful for biosensing as well as its ability to induce apoptosis, lending it to applications in drug delivery. UV-visible absorption spectroscopy was used to quantitate the presence Cyt C on AuNPs functionalized with ligands of varying electrostatic charge. Also, the quantitation of the presence of monolayers or multilayers was important to know when considering the concentration of drugs or biomolecules conjugated to AuNPs as a multilayer may interfere with their function, leading to an inaccurate representation of drug/biomolecule delivery or activity.