Rubifolic acid is primarily used in pharmaceutical research due to its structural similarity to folate compounds, making it a potential candidate for studying cellular metabolism and enzyme inhibition. It has shown interest in oncology research, particularly in the development of antitumor agents, as it may interfere with folate-dependent pathways crucial for cancer cell proliferation. Additionally, rubifolic acid is investigated for its role in modulating one-carbon transfer reactions, which are essential in DNA synthesis and repair. Its application extends to biochemical assays where it serves as a reference compound or inhibitor in studies involving folate metabolism. Due to its bioactive properties, it is also explored in the context of anti-inflammatory and antimicrobial agent development, although such uses remain largely experimental.