Amphiphilic graft copolymer consisting of poly(γ-glutamic acid) (γ-PGA) as the hydrophilic backbone and L-phenylalanine ethyl ester (Phe) as the hydrophobic side chain is an important biodegradable polymer with great potential in medical applications. In this research, we established analytical methods for the characterization and quality control of γ-PGA-graft-Phe (γ-PGA-Phe), which forms nanoparticles in aqueous solution, as a deployment platform in practical applications for vaccine adjuvants. The SEC-RI/MALS system, which uses size exclusion chromatography (SEC) coupled with a multi_angle light scattering (MALS) detector and refractive index (RI) detector, was developed to evaluate the characteristics of various types of polymers. By this method, it was indicated that absolute molecular weight (MW) should be used to measure the branch polymer. A gradient reversed phase HPLC (RP-HPLC) method was developed for the content of γ-PGA-Phe and the impurity levels to control product quality and safety. This quantitative approach could become key elements for identifying and characterizing γ-PGA-Phe. In addition, the degradation mechanism of γ-PGA-Phe was also identified as cleavage of main-chain of γ-PGA-Phe based on the stability study of γ-PGA-Phe in buffer solution with various pH values. The analytical developments described above will be important for use in both characterization and formulation design of biopolymers. Nanoparticles (NPs) composed of well-characterized biodegradable γ-PGA-Phe are expected to have a variety of potential clinical applications such as their use as drug and vaccine carriers.