Lipidation at the N-terminal of recombinant outer surface protein A (rOspA) is an important determinant of its immunogenicity. Lipidation patterns of rOspA can be sensitive to processing environments and storage conditions. In order to assure product consistency and stability. it is essential to characterize and monitor lipidation patterns of rOspA through its life-cycle. Electrospray mass spectroscopy combined with maximum entropy calculation was employed to analyze the lipidation of rOspA. The results revealed that more than 90% of protein is a tri-lipidated rOspA and the remainder is di-lipidated. It was demonstrated that the method is both sensitive and quantitative and has the potential to be used for routine quality control and stability testing.