Bioluminescence, the emission of light by living organisms, results from chemiluminescent reactions facilitated by enzymes like luciferases. Among these, NanoLuc (NLuc) stands out due to its exceptional brightness, stability, and compact structure, making it a valuable tool in bioassays and imaging applications. NLuc is a 19.1 kDa monomeric enzyme derived from the deep-sea shrimp Oplophorus gracilirostris. Its structure comprises eleven antiparallel β-strands forming a β-barrel, capped by four α-helices. To enhance its versatility, NLuc has been engineered into split forms. The two-component split system divides NLuc into a large fragment and a small peptide, which can be either low-affinity (SmBiT) or high-affinity (HiBiT). Building upon this, a three-component system incorporates an additional peptide, offering improved control and potential applications in chemical biology. Despite the advancements in split NLuc applications, several impediments exist that can be addressed to improve these systems. Recent studies have illuminated the allosteric mechanisms of NLuc. The enzyme exhibits homotropic negative allostery, where product binding to an allosteric site inhibits substrate binding at the catalytic site. Ongoing research into its structural dynamics and allosteric behaviors continues to expand its potential applications, while efforts to enhance the efficiency of its split forms aim to broaden its utility in complex biological assays. In this in silico assay, we clarify the sources of malfunctions in split NLuc and also explore aspects of split NLuc technologies. We examine some hypotheses of NLuc mechanisms that display the complex behavior of this luciferase.