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Aims: The aim of this study was to construct the improved pMAL expression vector to increase the efficacy of purification of small native peptides and their clear‐cut separation from MBP tag. The modifications we introduced can be applied to many expression vectors. Methods and Results: To improve the pMAL expression vector, we introduced the His6 tag and the enterokinase cleavage site (Ek) downstream from the MBP tag and Xa cleavage site on the original vector. For cloning of a desired peptide DNA, the enterokinase site contains a unique BsaBI restriction site adjacent to the original multi‐cloning site. This redesigned pMAL vector was optimized for the purification of cytoplasmic (pMALc5HisEk) and periplasmic (pMALp5HisEk) peptides. The purification of native and active peptide (P) was obtained following two‐step affinity chromatography. In the first step, the entire MBP‐His6‐Ek‐P fusion protein is purified using the Ni‐NTA agarose column. This fusion protein was cleaved with active His6 tagged enterokinase. In the second step, the further purification was performed by column containing the mixture of amylose and Ni‐NTA agarose resins. This removes both the MBP‐His6 and His6‐enterokinase leaving pure native protein in solution. These new vectors and the two‐step purification protocol were successfully applied in purification of active native small antimicrobial peptides (AMPs), lactococcin A and human β‐defensin. Conclusions: We constructed the improved pMAL expression vectors and established the pipeline and optimal conditions for their use in efficient purification of large amounts of active native small peptides. Significance and Impact of the Study: Choice of expression vector impacts on the efficiency of expression and purification of desired proteins. The idea of redesigning pMAL vector was driven by the need for rapid purification of larger amounts of active native AMPs. This newly improved pMAL vector, the cloning strategy, expression conditions and two‐step purification protocol represent a unique simple approach which can be applied in every laboratory. [ABSTRACT FROM AUTHOR]