Detection of genotypes and drug resistance mutations are important molecular tools assisting in clinical management of patients with chronic hepatitis B and C. Together with methods for assessment of genetic heterogeneity and relatedness of viral strains, they form the foundation of molecular surveillance. Currently, all these methods are based mainly on DNA sequencing followed by phylogenetic analysis. Mass spectrometry (MS) emerged recently as a rapid, cost-effective, reproducible and accurate alternative approach. MS-based molecular assays are highly amenable to automation and provide a suitable platform for routine application to the surveillance of HBV and HCV infections.