Autotransporters (ATs) are a superfamily of bacterial virulence factors on the bacterial outer membrane that are only produced by Gram-negative bacteria. Once an autotransporter is translocated across the inner membrane, its β-barrel domain folds and is inserted into the outer membrane, forming a secretion pore that is required to translocate the passenger domain to the bacterial cell surface. Our recent findings have shown that extracellular loop 5 (L5) of the β-barrel domain is essential for the correct folding of the passenger domain of autotransporters Pet and EspP, which belong to the SPATE (Serine Protease Autotransporters of Enterobacteriaceae) subfamily of autotransporters. In this current study, we aimed to investigate whether the role of L5 is conserved in other SPATEs using EspC from enteropathogenic Escherichia coli and Tsh from avian pathogenic E. coli as model SPATE proteins. By monitoring the assembly of EspC and Tsh variants carrying modifications in the L5 β-hairpin in live bacterial cells, we provide evidence that L5 is required for folding of these SPATE passenger domains into a stable β-helix. This suggests that L5-assisted folding of the passenger domain represents a conserved folding mechanism for SPATE autotransporter proteins. Future biophysical studies on refolded protein will determine if the nucleation of passenger domain folding can occur independently of the cellular machinery such that it is entirely mediated by the β-barrel domain. Since the passenger domain of SPATEs are often linked to the pathogenesis of many Gram-negative bacteria, L5 can serve as a potential target for the design of novel antibacterial drugs to treat a range of infectious diseases.