Lassa virus (LASV) remains a persistent threat to public health, and to combat this, various therapeutics have been developed, but their effectiveness is limited due to the virus's strain variability. Therefore, mutation‐based reverse vaccinology approaches were implemented to formulate an epitope‐based vaccine against the LASV, considering the variability in the glycoprotein. The glycoprotein was examined to screen out the B and T cell epitopes and further examined for the immunodominant epitope activity assessment. These epitopes were mapped with the identified position to introduce variability. 2 LBL (Linear B‐cell lymphocyte), 21 MHC‐I (Major Histocompatibility Complex Class I), and 8 MHC‐II potential epitopes were considered for wild and mutated (based on the mutation mapping). The wild and mutated vaccines were separately constructed, which comprise 545 amino acids in length by adjoining B and T cell epitopes via a specific linker, and also an adjuvant, PADRE epitope, 6xHis‐Tag, was incorporated to enhance the effectiveness. The formulated vaccine showed acceptable 3D structure quality (most favoured of wild: 91.5% and mutated: 91%) and high population coverage, i.e., 94%. The docking examination of wild and mutated vaccine with toll‐like receptor 2 (TLR‐2) revealed strong binding affinity, that is, −11.1 and −19.9 kcal/mol, and remarkable stability over 100 ns simulation based on the RMSD, RMSF. The immune simulation and in silico‐assisted cloning demonstrated a robust immune response and a remarkable expression in Escherichia coli system based on the GC% (wild; 57.51 and mutated; 57.57) and similar codon adaptive index value, that is, 0.93. The integrated approach will certainly aid in designing a mutation‐based epitope‐based vaccine that may counter different strains of LASV.