A single-stranded DNA-binding protein (SSB) has been recently proven to facilitate highly efficient separation of the excess hybridization probe from the probe-target hybrid in gel-free capillary electrophoresis. SSB added to the electrophoresis run buffer binds the single-stranded DNA probe but does not bind the double stranded DNA-DNA or DNA-RNA hybrid. As a result, SSB changes the electrophoretic mobility of the probe but does not affect the mobility of the hybrid. If the probe is labeled fluorescently, real-time sensitive detection can be facilitated. While the concept of SSB-mediated hybridization analysis has been proven in principle, the question of how to make such analysis quantitative without building a calibration curve remains open. Here, we propose a general approach for making SSB-mediated analysis quantitative. This approach takes into account such phenomena as (i) the potential influence of the probe-target hybridization and probe-SSB binding on the quantum yield of the fluorescent label and (ii) the potential dissociation of the hybrid by SSB. The proposed approach was used to study the quenching and the dissociation phenomena experimentally. We proved for the first time that SSB does not detectably dissociate the probe-target hybrid, which significantly simplifies the analysis.