The article introduces CAT-S, a bioorthogonal photocatalytic chemistry-enabled proximity labeling method for in situ profiling of mitochondrial proteomes in primary living samples. CAT-S uses a thioquinone methide (thioQM) labeling probe and an iridium-based photocatalyst to enable high-efficiency and specific labeling of mitochondrial proteins in living cells. The method is non-genetic, non-toxic, and applicable to a wide range of primary living samples, including diverse cell cultures, dissociated mouse tissues, and primary T cells from human blood. CAT-S has been applied to various samples, revealing novel mitochondrial proteins and uncovering hidden mitochondrial functions. It also enables the quantification of proteomic perturbations in dysfunctional tissues, such as diabetic mouse kidneys, providing insights into disease mechanisms. The method's advantages include its ability to capture subcellular proteomes in native states, its high spatiotemporal resolution, and its non-genetic operation, making it a valuable tool for subcellular proteomic investigations in primary samples.The article introduces CAT-S, a bioorthogonal photocatalytic chemistry-enabled proximity labeling method for in situ profiling of mitochondrial proteomes in primary living samples. CAT-S uses a thioquinone methide (thioQM) labeling probe and an iridium-based photocatalyst to enable high-efficiency and specific labeling of mitochondrial proteins in living cells. The method is non-genetic, non-toxic, and applicable to a wide range of primary living samples, including diverse cell cultures, dissociated mouse tissues, and primary T cells from human blood. CAT-S has been applied to various samples, revealing novel mitochondrial proteins and uncovering hidden mitochondrial functions. It also enables the quantification of proteomic perturbations in dysfunctional tissues, such as diabetic mouse kidneys, providing insights into disease mechanisms. The method's advantages include its ability to capture subcellular proteomes in native states, its high spatiotemporal resolution, and its non-genetic operation, making it a valuable tool for subcellular proteomic investigations in primary samples.