Numerous biotechniques rely on the use of split-fluorescent proteins (split-FPs) to visualize protein-protein interactions, to fluorescently label cells, to assemble nanomaterials or to study the nanoscale dynamics of individual biomolecules in vivo. Surprisingly little is known about the reassembly mechanisms of split-FPs and there is a lack of characterization concerning their biochemical and biophysical properties including fragment binding rates, folding and chromophore maturation rates and overall FP brightness following the interaction of complementary peptide fragments. Through targeted mutagenesis we have developed a variety of self-complementing and asymmetric split-FPs (sGFP, sYFP, sCFP) and characterized their in vitro and in vivo photophysical and biochemical properties using fluorescence kinetic measurements, fluorescence anisotropy, cell imaging, and single molecule biophysical techniques. We show that, in vitro, split-FP complementation follows a conformational selection mechanism whereby the larger split-FP fragments exist in a monomer-dimer equilibrium and only monomers are competent for binding the smaller peptide fragments. This bimolecular interaction involves a slow and irreversible binding step of the peptide followed by a fast maturation step of the FP chromophore. Interestingly, under large excess of the small peptide fragment, split-FPs populate an intermediate state and the complementation proceeds via a different reaction pathway involving a conformational change of the larger split-FP fragment. Based on these in vitro characterizations, the brightest and the fastest maturing split-FPs were expressed as fusion proteins in cells to assess their biochemical behaviors in vivo and their respective performance for ensemble and single molecule cellular imaging. This study resulted in development of novel split-GFP and split-YFP variants with improved maturation kinetics, brightness, and photostability for Complementation Activated Light Microscopy (CALM) imaging of cellular processes.