The synthesis of several novel poly(phenyleneethynylene)s (PPEs) functionalized with different natural α-amino acids methyl esters has been achieved through Cassar-Heck-Sonogashira reaction. Five different derivatives have been prepared varying the nature of the amino acid (Gly, Leu, N-methyl Leu, Phe, and Val), and their aggregation behavior has been investigated by means of UV-vis absorption, circular dichroism (CD), and fluorescence spectroscopies in different conditions of aggregation. ECD measurements provided unique information about the structural organization of the aggregates dispersed in solution and as thin films. The effects of the nature of the amino acidic moiety, the consequences of chirality, and the role played by intermolecular hydrogen bonds have been elucidated.