For a comprehensive exploration of the Bimolecular Fluorescence Complementation (BiFC) approach, we critically assess whether protein pairs establish direct or indirect interactions within this context. Emphasizing the multifaceted nature of protein interactions, this review underscores the imperative of delineating macromolecular complex characteristics across five distinct levels: composition, stoichiometry, copy number, topology, and dynamics. A pivotal discussion revolves around the challenges introduced by the overexpression of target proteins and the potential artificial ramifications of fusion proteins. The significance of stoichiometry in protein interactions is illuminated, with the HOXA9/PBX1 interaction serving as a pertinent case study. The research identifies and elaborates on the inherent limitations of prevailing large-scale BiFC screenings, advocating for the incorporation of rigorous negative controls. A novel proposition is introduced in the form of a high-throughput multicolor BiFC strategy, exemplified using the HOXA9 and PBX1 proteins. The paper culminates with an insightful discourse on the implications of tags within the BiFC system, presenting the TriFC (Tripartite Fluorescence Complementation) assay as a promising alternative.