Abstract Body: Background. Mitochondrial intermembrane space (IMS) proteases regulate protein quality control, trafficking, mitophagy, apoptosis, and mitochondrial dynamics to maintain mitochondrial integrity and prevent proteotoxic stress. Dysregulated IMS proteases are linked to cardiovascular and metabolic diseases. Understanding their regulation and interdependence is key to identifying compensatory mechanisms that sustain mitochondrial function and potential therapeutic targets. Hypothesis. IMS proteases function within an interconnected network, where the loss of individual proteases induces compensatory changes affecting mitochondrial function. Specifically, IMS protease knockouts alter other proteases expression and impact stress response pathways. Aims. This study identifies compensatory mechanisms maintaining mitochondrial homeostasis following IMS protease loss and assesses their impact on mitochondrial function. Methods. CRISPR/Cas9 was used to generate IMS proteases knockouts (NLN, HTRA2, LACTB, YME1L1, OMA1, ATP23, PARL, IMMP1L, IMMP2L, LACTB2) in the HEK293 cells. Immunoblotting analyzed compensatory changes and interdependencies. RNA sequencing evaluated transcriptomic shifts. Functional assays, including mitochondrial calcium retention capacity (CRC), calcium flux, and oxygen consumption rate (OCR) analysis, assessed mitochondrial performance. Results. PARL was upregulated in most knockouts, indicating its adaptive role, while IMMP2L expression was consistently reduced, suggesting dependence on other IMS proteases. Knockouts induced broad transcriptomic and functional changes, particularly in oxidative stress, apoptosis, and protein quality control, suggesting compensatory responses. Functional assays revealed mitochondrial dysfunction, including decreased CRC and OCR along with deregulated calcium flux, underscoring the critical role of IMS proteases in mitochondrial homeostasis. Conclusion(s). IMS proteases operate within a tightly regulated network where their loss disrupts mitochondrial homeostasis and activates compensatory mechanisms. Understanding these interactions may reveal novel therapeutic strategies for mitochondrial diseases.