Amphiphilic copolymers of hydrophobic Disperse Red 1 acrylate (DR1A) and hydrophilic acrylic acid (AA) were dissolved in THF and a H2O/THF cosolvent series. The thermal cis-to-trans isomerization behavior of the azo groups was measured by means of flash photolysis to investigate self-assembled structure in solution. The rate constants observed for the azo-containing polymers permit an estimation of how the thermal isomerization is influenced by both polymer and solution properties. In pure THF the relaxation was observed to be the usual monoexponential decay. In mixed aqueous-organic solvent solutions however, all poly(DR1A-co-AA) samples showed a highly unusual biexponential rate decay, with both a slow isomerization process due to inhibited mobility of the azo groups, and a faster isomerization process for unaggregated azo groups. In these systems, the relaxation of the cis-isomer was fit well with a biexponential function, which clearly demonstrates two distinct local chromophore environments. The spectroscopic results of this study suggest that weakly aggregated states of the amphiphilic polymers in an aqueous-organic solution can be readily studied through the simple technique of isomerization spectroscopy to estimate the fractions of aggregated groups involved and their local environment.