Background and Purpose The calcium ion (Ca²⁺) is a versatile cellular messenger regulating various biological processes. Compounds modulating subcellular Ca²⁺ signals hold substantial pharmacological potential. Advances in fluorescent biosensors have revolutionized Ca2+ imaging. Still, co-expression of targeted biosensors for simultaneous measurement of Ca2+ signals in multiple cellular compartments is complicated by heterogeneous expression levels of the various sensors. Experimental Approach Here, we introduce CARMEN, a ribosomal skipping-based quad-cistronic fluorescent biosensor system that enables high-content Ca²⁺ imaging across three compartments. CARMEN facilitates equal co-expression of spectrally distinct Ca²⁺ biosensors: the near-infrared Ca²⁺ biosensor for the cytosol (NIR-GECO2G-NES), the green Ca²⁺ biosensor for mitochondria (CEPIA3mt), the red Ca²⁺ biosensor for the endoplasmic reticulum (R-CEPIA1er), along with a Ca²⁺-insensitive blue fluorescent protein targeted to the nucleus (NLS-mTagBFP2), serving as a normalization reference. Key Results CARMEN allows spatiotemporal correlation of Ca²⁺ signals across the cytosol, ER, and mitochondria, revealing distinct dynamics. We noted delayed mitochondrial Ca²⁺ uptake compared to the other compartments. We validated CARMEN across three cell types and tested two recently identified mitochondrial Ca²⁺ uniporter inhibitors (MCUis), MCUi4 and MCUi11, showcasing the potential of CARMEN for its application in pharmacological research. Our results show that while both MCUi4 and MCUi11 inhibited mitochondrial Ca²⁺ uptake in HeLa S3 cells, MCUi4 reduced cytosolic Ca²⁺ signals and oscillations, whereas MCUi11 had opposing effects. Conclusions and Implications CARMEN is a powerful tool for real-time, multiplexed analysis of compartment-specific Ca²⁺ signals, with the potential for automation in high-content drug screening.