Membrane-active pore-forming α-helical peptides and proteins are well known for their dynamic assembly mechanism and it has been critical to delineate the pore-forming structures in the membrane. Previously attempts have been made to elucidate their assembly mechanism and there is a large gap due to the dynamic, complex and varied pathways by which these membrane-active pores impart their effect. Our work attempts to dissect the assembly pathway and potential applications of simplified synthetic peptide pores based on naturally occurring transmembrane pores. In this work, we demonstrate the multi-step structural assembly pathway of α-helical peptide pores formed by a 37 amino-acid synthetic peptide based on the natural porin from Corynebacterium urealyticum using single-channel electrical recordings. More specifically, we report a detectable intermediates during membrane insertion of alpha-helical peptides. The fully assembled pore is functional and exhibited unusually large stable conductance and voltage-dependent gating, generally applicable to a range of pore-forming proteins. Furthermore, we used rationally designed mutants to understand the role of specific amino acids in the assembly of these peptide pores. We suggest that our study contributes to understanding the mechanism of action of alpha-helical pores and antimicrobial peptides and should be of broad interest to bioengineers to build peptide-based nanopore sensors.