It is extremely important for pharmaceutical formulators to have analytical methodology that provides efficient detection and quantification of HPβCD aggregates. Five different methods were then evaluated for their potential to detect these aggregates and to determine critical aggregation concentration (cac): osmometry, viscometry, tensiometry, dynamic light scattering (DLS), and permeability studies. Overall, tensiometry was an inadequate method with which to study HPβCD aggregation, since the addition of HPβCD to water resulted in only minor changes in surface tension. Osmolality and viscosity studies have shown that for HPβCD, solute–solvent interactions are the main contributors for the observed deviation from ideality. These deviations might be related to the presence of aggregates. The DLS method proved to be an effective method with which to detect HPβCD aggregates and estimate their hydrodynamic diameter, although it presented some limitations concerning their quantification. In terms of the assessed methods, permeation studies were shown to be the best to study HPβCD aggregation phenomena, since they were the only method where the detection of aggregates and the determination of apparent cac values was possible. Also, it was the least invasive for the HPβCD samples and the method that provided more conclusive data. Results suggested that HPβCD, as expected, has less tendency to form aggregates than βCD.