Bimolecular fluorescence complementation (BiFC) was introduced a decade ago as a method to monitor alpha-synuclein (alpha-syn) oligomerization in intact cells. Since then, several alpha-syn BiFC cellular assays and animal models have been developed based on the assumption that an increase in the fluorescent signal correlates with increased alpha-syn oligomerization or aggregation. Despite the increasing use of these assays and models in mechanistic studies, target validation and drug screening, there have been no reports that (1) validate the extent to which the BiFC fluorescent signal correlates with alpha-syn oligomerization at the biochemical level; (2) provide a structural characterization of the oligomers and aggregates formed by the BiFC. To address this knowledge gap, we first analysed the expression level and oligomerization properties of the individual constituents of alpha-syn-Venus, one of the most commonly used BiFC systems, in HEK-293 & SH-SY5Y cells from three different laboratories using multiple biochemical approaches and techniques. Next, we investigated the biochemical and aggregation properties of alpha-syn upon co-expression of both BiFC fragments. Our results show that (1) the C-terminal-Venus fused to alpha-syn (alpha-syn-Vc) is present in much lower abundance than its counterpart with N-terminal-Venus fused to alpha-syn (Vn-alpha-syn); (2) Vn-alpha-syn exhibits a high propensity to form oligomers and higher-order aggregates; and (3) the expression of either or both fragments does not result in the formation of alpha-syn fibrils or cellular inclusions. Furthermore, our results suggest that only a small fraction of Vn-alpha-syn is involved in the formation of the fluorescent BiFC complex and that some of the fluorescent signal may arise from the association or entrapment of alpha-syn-Vc in Vn-alpha-syn aggregates. The fact that the N-terminal fragment exists predominantly in an aggregated state also indicates that one must exercise caution when using this system to investigate alpha-syn oligomerization in cells or in vivo. Altogether, our results suggest that cellular and animal models of oligomerization, aggregation and cell-to-cell transmission based on the alpha-syn BiFC systems should be thoroughly characterized at the biochemical level to ensure that they reproduce the process of interest and measure what they are intended to measure.