The intracristal space (ICS) is a distinct subdomain of the mitochondrial intermembrane space (IMS) where oxidative phosphorylation occurs. This region supposedly controls the dynamic demands regulating mitochondrial respiration; however, its other molecular components remain largely unknown. To remedy this, we developed isotope-coded desthiobiotin-phenol probes that enabled differential mass analysis of post-translocated modifications. Using this unique proximity labelling approach dubbed “iSpot-ID”, we discovered that TMEM177 is exclusively localised to the ICS and that the protein offers an ICS targeting modality for studying the unique characteristics of ICS, such as the local pH, redox states, and temperature in the space. Furthermore, we obtained a proteome specifically localised to the ICS using iSpot-ID with TMEM177-APEX2, which included a high portion of mitochondrial matrix-targeting proteins and several known IMS-localised proteases as well as subunits of the OXPHOS complex. This protein accumulation was enriched by inhibiting the import of mitochondrial matrix proteins or by inducing other stress conditions; the protein was consequently degraded by proteases in the ICS. Our data revealed that the ICS is important for mitochondrial quality control under dynamic cellular states.