For regulatory approval, the comparability of a biosimilar product to an originator product should be ensured through thorough physicochemical and biological characterization. To evaluate the biosimilarity between LBDE, the proposed biosimilar darbepoetin alfa, and NESPA (R), its originator, we performed a comprehensive physicochemical and biological characterization study. Primary and higher-order protein structures were analyzed using Lys-C peptide mapping with liquid chromatography-mass spectrometry (LC-MS), disulfide bond identification, circular dichroism, and fluorescence spectroscopy. Glycosylation and isoform distribution were analyzed using MS, LC, and capillary zone electrophoresis. Size variants were evaluated with size-exclusion chromatography-high-performance liquid chromatography (SEC-HPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Biological characterization included binding affinity for human erythropoietin receptor, in vitro cell proliferation, and in vivo potency. Pharmacokinetics (PK) were evaluated using rats through two injection routes. Non-reducing and reducing Lys-C peptide mapping showed a highly similar peak profile, confirming that LBDE and NESPA (R) have the same primary structure and disulfide bonds. Glycosylation and isoform analyses showed that the attached N-glycan and O-glycan structures were the same and their relative contents were similar. Spectroscopic analysis of LBDE showed indistinguishable spectra with NESPA (R). For both LBDE and NESPA (R), a very small amount of size variants was found in SEC-HPLC, and no minor bands were detected in SDS-PAGE. Furthermore, LBDE did not show any difference with NESPA (R) in the in vitro and in vivo functional analyses. PK parameters of LBDE were in good agreement with those of NESPA (R). LBDE shows high similarity to NESPA (R) with regard to structure and function.