Amphiphilic porphyrins are of great interest in the field of supramolecular chemistry because they can be fabricated into highly ordered architectures that are stabilized by π-π stacking of porphine rings as well as by non-covalent interactions between their hydrophilic substituents. Protoporphyrin IX (PPIX) has two flexible propionic acid tails and is one of the most common amphiphilic porphyrins. However, unlike other PPIX analogues, PPIX does not form stable extended nanostructures, and the reason for this is still not understood. Here, we employ ion mobility mass spectrometry in combination with infrared multiple photon dissociation spectroscopy to investigate early aggregates of PPIX. The ion mobility results show that growth occurs via single-stranded face-to-face stacking of PPIX. From the infrared spectroscopy on well-defined aggregates, it can be concluded that pairing of the carboxylic acid groups of the tails is a stabilizing element and that such a pairing occurs across a third residue from residue n to residue n+2. The tetramer appears to be especially stable, because all of its propionic acid tails are optimally paired and no free tails to promote further growth are present, which possibly prevents PPIX from forming larger, well-ordered assemblies.