The biochemical, biophysical and functional characterization of transmembrane β-barrels of human and bacterial origin has provided insight on factors that stabilize these proteins in various lipid and detergent systems. This review attempts to summarize our recent findings and contributions to the field of membrane protein biophysics. Using various β-barrels, particularly, the 8-stranded OmpX and Ail from Escherichia coli and Yersinia pestis, respectively, and the 19-stranded human mitochondrial transmembrane barrel VDAC-2 (the voltage-dependent anion channel isoform 2) as model systems, we have observed that barrel-micelle interaction strengths, protein-to-detergent ratios and interface residues serve as key elements in determining the stability of these barrel systems, irrespective of the protein origin. Differences in the behaviour of these barrels arise from their malleability to reversible folding versus stabilization by kinetic contributions, with the latter giving rise to irreversible unfolding and aggregation. Furthermore, while bacterial proteins are more tolerant to mutations, hVDAC-2 is particularly influenced by subtle (conserved) mutations, both in its stability and function. The implications of these findings to the field of membrane proteins are also discussed.