In summary, we have developed a new "off-on" fluorescent probe (RhQ) for the selective detection and for visualizing of the dynamics of VDPs in different types of live cell lines. Simultaneous fluorescence imaging using RhQ provided the detailed localization of VDPs in whole live cells. Interestingly, live cell imaging with RhQ clearly indicated the presence of cell surface protein vicinal dithiols on MCF-7 cells, while no cell surface protein vicinal dithiols were labelled by RhQ on CHO-K1 cells. The colocalization experiments further indicated that the most abundant VDPs localized in the mitochondria of live MCF-7 and CHO-K1 cells. Significantly, the proteomic analysis of the MCF-7 cell lysis unveiled some interesting and functional cellsurface VDPs. As such, RhQ could be used as an effective tool for the tracing of VDPs in live cells. The approach of long-term dynamic tracing based on time-lapse imaging with RhQ helped map the distribution of endogenous protein vicinal dithiols in live cells. The fluorescence released from RhQ also paints an overall picture of the changes in cellular protein vicinal dithiol profiles in different live cell lines. Thus, by using RhQ based long-term dynamic tracing technology and proteomics, new insights into the location of endogenous protein vicinal dithiols can be obtained, which we hope will fuel enthusiasm into research surrounding the role of protein vicinal dithiols in the regulation of diseases and cellular uptake, as well as promote improved designs for cell surface thiol-based drug candidates and vectors.