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The colloidal structure of asphaltenes impacts various physical properties and is important to characterize. Previously, in both laboratory and oilfield studies, asphaltenes have been shown to form nanoaggregates. In addition, previous work has shown that asphaltenes exhibit a critical nanoaggregate concentration (CNAC) in toluene in the range of 50−150 mg/L. In this study, centrifugation is used to prove a major change of asphaltene aggregation at the CNAC concentration, thereby corroborating previous results. Collection of these nanoaggregates by centrifugation validates there existence. The nanoaggregate size is found to be ∼2.5 nm, which is compatible with corresponding previous determinations from gravitational gradients. Asphaltene monomers are seen to be small (<1.5 nm), confirming previous diffusion measurements and corroborating the now common view that asphaltene molecular size is rather small. A two-component, monomer and nanoaggregate, phase equilibrium model is shown to treat the primary features of the data; nevertheless, shortcomings of this model are discussed. These centrifugation experiments are simple and we believe compelling confirmation of the asphaltene CNAC in toluene.