Large serine integrases (LSIs) offer tremendous potential for rapid genetic engineering as well as building biological systems capable of responding to stimuli and integrating information. Currently, there is no unified metric for directly measuring the enzymatic characteristics of LSI function, which hinders evaluation of their suitability to specific applications. Here, we present an experimental protocol for recording DNA recombination in HEK293 cells in real‐time through fluorophore expression and software which fits the kinetic data to a model tailored to LSI recombination dynamics. Our model captures the activity of LSIs as three parameters: expression level (Kexp), catalytic rate (kcat), and substrate affinity (Kd). The expression level and catalytic rate for phiC31 and Bxb1 varied greatly, suggesting disparate routes to high recombination efficiencies. Moreover, the expression level and substrate affinity jointly impacted downstream reporter expression, potentially by obstructing transcriptional machinery. We validated these observations by swapping between promoters and mutating key recombinase residues and DNA recognition sites to individually modulate each parameter. Our model for identifying key LSI parameters in cellulo provides insight into selecting the optimal recombinase for various applications as well as for guiding the engineering of improved LSIs. Large serine integrases are a group of site‐specific recombinases that can modulate gene expression in living cells and report the transition through fluorescence, making them a potentially useful tool in a range of biological applications. However, endpoint readouts of fluorescence do not capture the whole story, as gene expression, protein‐DNA interaction, and recombination catalysis all factor into the rate and magnitude of the observed phenotypes. Here, George Church et al. present an experimental protocol that integrates fluorescence with DNA and protein concentrations and apply this model to identify three key parameters that can encapsulate the dynamics of the recombination process.