Understanding how cells maintain a functional proteome and respond to stress is crucial for deciphering molecular pathogenesis and developing treatments for diseases such as neurodegenerative disorders. Achieving finer quantification of cellular proteostasis efficiency, phase transitions, and local environmental changes remains a key but challenging goal. Here, we present our recent progress in developing fluorescence-based strategies and methodologies to study proteostasis. To assess proteostasis efficiency, we directly measure the unfolded protein load in cells, as a decline in proteostasis capacity leads to the accumulation of unfolded proteins. To this end, we designed and synthesized a series of cysteine-reactive fluorogenic probes that selectively react with unfolded proteins exposing cysteine residues, producing a fluorescence turn-on response. Remarkably, these probes exhibit strong fluorescence with unfolded proteins, weak fluorescence with folded proteins containing surface cysteines, and no fluorescence with small-molecule biothiols. We applied this method to monitor proteostasis collapse in cells exposed to pharmacological stressors, expressing mutant proteins in Huntington’s disease models, or infected with viruses. When integrated with mass spectrometry-based proteomics, our approach also enables the identification of basal intrinsically disordered proteins and those that become unfolded upon stress. Together, this strategy provides a powerful platform for probing protein unfolding and stress responses and holds promise for biomarker discovery and therapeutic development.