A series of arborescent poly(L-glutamic acid)s of generations 0 to 3 (PGA(GY) with Y = 0-3) were randomly labeled with 1-pyrenemethylamine to yield several Py-PGA(GY) constructs with pyrene contents ranging between 2.5 and 22 mol %. The density (rho) of the interior of the PGA(GY) samples was estimated in N,N-dimethylformamide (DMF) and dimethyl sulfoxide (DMSO) by conducting gel permeation chromatography and dynamic light scattering experiments to determine their molar mass and hydrodynamic diameter, respectively. It was determined that rho increased with the generation number from PGA(G1) to PGA(G2), which promoted more contacts between the pyrene labels. The increase in the number of pyrene-pyrene contacts was quantified with the parameter N-bl(ob) obtained by analysis of the fluorescence decays for the Py-PGA(GY) samples in DMF and DMSO. In the analysis, N-blo(b) represented the number of structural units, i.e., glutamic acid residues, comprised inside the volume probed by an exited pyrene, termed a blob. Inside a blob, pyrene excimer formation (PEF) could occur upon diffusive encounters between an excited- and a ground-state pyrene label. It was found that N-bl(ob) increased with the generation number, and larger N-bl(ob) values were retrieved in DMSO as compared to DMF because the oligo(L-glutamic acid) (OGA) side chains in the PGA(GY) samples underwent partial loss of helicity in DMSO, which increased their hydrodynamic volume and forced the side chains closer to each other, thus resulting in more pyrene-pyrene contacts and larger N-bl(ob) values. The trends observed for N-bl(ob) in DMF as a function of the generation number could be correlated theoretically with the degree of polymerization of the OGA side chains used to prepare the PGA(GY) samples and their internal density rho. The good agreement found between the theoretical and experimental N-bl(ob) values confirms that pyrene is an excellent probe to study the complex interior of partially structured polypeptides.