Ribulose-1,5-biphosphate-carboxylase/oxygenase (RuBisCO) from alfalfa is a promising source of alternative food protein, but it is challenging to purify. Ultrafiltration shows potential for concentrating and fractionating RuBisCO from low molecular weight compounds present in the extracted juice, but there is limited information on their partitioning depending on the membrane pore size. Therefore, the objective of this study was to evaluate the partitioning of various compounds from the juice depending on membrane pore size. It was hypothesized that both membrane pore sizes will retain RuBisCO but might partition low molecular weight compounds within the matrix differently. RuBisCO was fully retained by both the 300 and 100 kDa polyethersulfone (PES) membranes, while ash (29.7%–35.4% dry matter (DM)), non-protein nitrogen (2.38%–2.53% DM), and free N-terminals (10.13–10.34 mg glutamic equivalent/100 mg DM) were transmitted through both membranes. The retentates showed a distinct yellow color when filtering with 100 kDa membranes, but not when using 300 kDa membranes. Although no significant differences in total polyphenol permeation were observed through a spectrophotometric assay, a metabolomics screening analysis showed that the 100 kDa membranes had less transmission of the flavonoids apigenin, chrysoeriol, tricin, and tricin glycosides, which might contribute to the yellow color. These results reiterate the importance of measuring individual polyphenols using hyphenated techniques as opposed to spectrophotometric total reducing capacity. Sixteen saponins were identified and their permeation was lower in the 100 than the 300 kDa membranes, indicating a better potential for higher degree of protein refinement for the latter. This study demonstrates for the first time the difference in partitioning of the small molecular weight components, using PES membranes of similar chemistry but different size, during ultrafiltration of alfalfa juice.