To validate the high selectivity of RhQ for vicinal dithiols on VDPs, we evaluated the fluorescence response of RhQ towards a variety of amino acids including mono-thiol containing amino acids (or peptides) such as Cys, Hcy and GSH. No significant fluorescence changes were observed for RhQ solutions in the presence of 200 equivalents of these mono-thiol containing reagents, respectively (Figure ). Similarly, the oxidized form of BSA (oBSA, the vicinal dithiols of BSA were treated with hydrogen peroxide) only induced negligible fluorescence changes of RhQ compared to the fluorescence signal from the rBSA treated RhQ solution (Figure ). In contrast, the control probe (HC) exhibited no response to rBSA (Figure ), demonstrating the trivalent arsenic group of RhQ is required to ensure the selective interaction of RhQ with vicinal dithiols on VDPs. Then, an SDS-PAGE assay was conducted to confirm the high selectivity of RhQ to VDPs. Only the RhQ and rBSA loaded lane exhibited fluorescence, while the RhQ and oBSA loaded lane exhibited no fluorescence. The Coomassie brilliant blue (CBB) staining clearly indicated that the fluorescent band was due to the formation of rBSA-probe complex (Figure ). No fluorescence signal was observed from the HC and rBSA loaded lane due to the lack of trivalent arsenic in HC. All these results suggest that the high selectivity of RhQ was from the specific interaction between the cyclic dithiaarsane part of RhQ and the vicinal dithiols of rBSA. After the FRET effect was interrupted by vicinal dithiols, the VDPs can be specifically labelled and visualized through the recovered fluorescence from the rhodamine group of RhQ. Finally, we explored the dynamic tracing of protein vicinal dithiols using RhQ in live cells. Initially, the cell toxicity of RhQ was evaluated. A cell counting kit-8 (CCK-8) indicated that RhQ has no significant effect on the viability of the live MCF-7 cells at concentrations between 0 and 30 μM even when the incubation time is extended to 48 h (Figure ). On loading live cells with probe RhQ the fluorescence signal changes were recorded every 2 min using a confocal microscope. A gradual increase of fluorescence signal from the live MCF-7 cells was obtained (Figure and Movie S1a). In contrast, the control probe HC-treated cells exhibited negligible fluorescence signal changes (Figure and Movie S1b). Semiquantitative calculations of the fluorescence intensity from the cells further indicated that the fluorescence from the RhQ-treated cells increased as a function of time and then reached a plateau at 90 min (Figure ). All these results suggested the high selectivity of RhQ towards VDPs in live MCF-7 cells. Furthermore, an interesting phenomenon was observed where the fluorescence signal was initially observed in the MCF-7 cell membrane and then the intracellular fluorescence signal gradually developed as the time increased (Figure and).