Serpins are a unique breed of proteins due to their enzymatic mechanism. Two systems were closely monitored during fluorescent binding studies, the ACT-CHY along with the AT:TRY interaction. Four different conformational variants of each system were studied including the native, cleaved, latent and complex forms. Three different fluorescent dyes were used to identify the conformations including ANS, TNS, and bis-ANS. SI studies and protease assays utilizing both Suc-AAPF-pNA and L-BAPNA were instrumental in determining conformations along with gel electrophoresis studies. The hydrophobic dyes bound to the different serpins with varying KD and ΔFmax due to structural variations among the conformers and the complex. Both TNS and bis-ANS gave higher ΔFmax values than ANS. Bis-ANS gave significantly higher ΔFmax values for the ACT:CHY than the other conformations, while also exhibiting relatively low KD value. KD values for the bis-ANS complexes are relatively low when compared to other fluorophores. Bis-ANS is more specific for the AT system than either TNS or ANS. Bis-ANS displays a ΔFmax of 36 fold for the ACT:CHY complex, while TNS displays a 27 fold increase for AT:TRY system. Modulation studies using bis-ANS to alter the kinetics of latent ACT formation proved unsuccessful, suggesting that fluorescent dyes have little, if any effect on serpin variant formation.