Size exclusion chromatography (SEC) has been used extensively to characterize asphaltenes, crude oils, and petroleum products. These studies have provided significant insights into the nature and behavior of this material. This work presents a couple of new applications that use this technique. First, a new methodology is presented for determining the asphaltene content based on using a SEC column. This new method shows good repeatability, and the results correlate with the gravimetric standard method ASTM D6560. Second, we explored the SEC behavior of a series of different asphaltenes injected at low concentrations (10−500 ppm) using an evaporative light scattering detector. The analysis of the changes in relative areas of the peaks in the chromatogram revealed a break in the behavior between 50 and 100 ppm which could have been associated with the existence of a critical nanoaggregate concentration (CNAC). However, based on the presence of significant peaks (∼30−40% of the total area) corresponding to asphaltene aggregates even at concentrations as low as 10 ppm, this break cannot correspond to a CNAC, instead it might be associated with a transition to a different aggregation mechanism. This work also explores the SEC behavior of asphaltenes precipitated using different solvents from the same crude oil and shows how the aggregation is correlated to the solvent used to obtain the asphaltenes, while the aggregate sizes are related to the average characteristics of the molecules that precipitate using each solvent.