The serine protease inhibitor (serpin), plasminogen activator inhibitor-1 (PAI-1), is an important biomarker for cardiovascular disease and many cancers. It is therefore a desirable target for pharmaceutical intervention. However, to date, no PAI-1 inhibitor has successfully reached clinical trial, indicating the necessity to learn more about the mechanics of the serpin. Although its kinetics of inhibition have been extensively studied, less is known about the latency transition of PAI-1, in which the solvent-exposed reactive center loop (RCL) inserts into its central β-sheet, rendering the inhibitor inactive. This spontaneous transition is concomitant with a large translocation of the RCL, but no change in covalent structure. Here, we conjugated the fluorescent probe, NBD, to single positions along the RCL (P13-P5′) to detect changes in solvent exposure that occur during the latency transition. The results support a mousetrap-like RCL-insertion that occurs with a half-life of 1–2 h in accordance with previous reports. Importantly, this study exposes unique transitions during latency that occur with a half-life of ˜5 and 25 min at the P5′ and P8 RCL positions, respectively. We hypothesize that the process detected at P5′ represents s1C detachment, while that at P8 results from a steric barrier to RCL insertion. Together, these findings provide new insights by characterizing multiple steps in the latency transition.