A process design methodology is presented for the supercritical fluid fractionation of liquid mixtures in countercurrent columns, where internal reflux is generated by a thermal gradient at the top stage. In the proposed methodology, the process design is carried out using the temperature at the top stage (T1) in the same fashion as the external reflux ratio (L0/D), with respect to the number of theoretical stages (N) and the solvent to feed ratio (S/F). The study carried out on the fractionation of fish oil ethyl esters (FOEE) shows that the internal reflux process is a viable alternative to the external reflux process. In the internal reflux process, optimal results are attained operating the isothermal section of the column at temperatures not above 50 ◦C. In this way, the internal reflux in the enriching section can be varied to a large extent without subjecting the oil to high temperatures at the top stage of the column. A direct comparison between internal and external reflux process is showed, considering the same feed and the same process specifications (mass fraction and recovery of heavy compounds in the raffinate equalto 95%) of a previous work focusing on the external reflux process [15]. The results on the internal reflux process demonstrate that the specifications can be attained operating the column at lower temperature (50 ◦C vs. 70 ◦C) and lower pressure (13.3 MPa vs. 16.7 MPa), but similar values of N and S/F. The minimum number of theoretical stages is approximately 10 in both cases. The minimum external reflux was found to be approximately 2.3 [15], whereas the minimum reflux conditions in the internal reflux process are found operating the top stage at approximately 64 ◦C.