The interest in lactobionic acid (LA) in nano-pharmaceuticals is growing due to its selective affinity to liver cells. Currently there is no suitable analytical method for the determination of LA in nanoparticles or other matrices. There is a need for a quantitative assay to accurately determine LA in nano-particles as well as in other matrices. In this study, a HILIC-MS/MS method was developed and validated for the analysis of LA. The method was applied for the assessment of chitosan nanoparticles (CNP) functionalization. The developed method was valid for the determination of LA with a LOD and LOQ of 1 and 5 ng/mL, respectively. The method was applied for the determination of unreacted LA after nanoparticle functionalization and revealed that a loading-capacity of 24 % was achieved. In conclusion, the developed method is the first report for the quantitative determination of LA functionalization on nanoparticles. The method can be directly applied in the quality-control applications for the assessment of nanoparticle functionalization with LA and may be extended for quality control applications of other LA-containing products.