Even though in the last few years several families of eukaryotic β-barrel outer membrane proteins have been discovered, their computational characterization and their annotation in public databases are far from complete. The PFAM database includes only very few characteristic profiles for these families, and in most cases, the profile hidden Markov models (pHMMs) have been trained using prokaryotic and eukaryotic proteins together. Here, we present for the first time a comprehensive computational analysis of eukaryotic transmembrane β-barrels. Twelve characteristic pHMMs were built, based on an extensive literature search, which can discriminate eukaryotic β-barrels from other classes of proteins (globular and bacterial β-barrel ones), as well as between mitochondrial and chloroplastic ones. We built eight novel profiles for the chloroplastic β-barrel families that are not present in the PFAM database and also updated the profile for the MDM10 family (PF12519) in the PFAM database and divide the porin family (PF01459) into two separate families, namely, VDAC and TOM40.