Natural Killer (NK) cells play a crucial role in the immune response against cancer, with ineffective killing leading to tumor growth. The two killing phenotypes, apoptosis and necrosis, cause distinct immunological impacts due to enhanced inflammatory responses upon necrosis. Understanding both the occurrence and phenotype of cancer cell killing is essential for assessing NK cell efficiency. Using the pCasper FRET sensor, coupling GFP and RFP with a caspase-cleaved linker sequence, viable, apoptotic, and necrotic cells can be distinguished. To automate analyses, we developed the MATLAB application MSparkles, to track cancer and killer cells, analyze and classify target cell death, and reconstruct the killing sequence of individual killer cells. Intracellular Ca²⁺ plays a pivotal role in the exocytosis of lytic granules. We compared different Ca²⁺ dyes regarding their phototoxicity during NK cell Ca²⁺ measurements, finding that Fura-10 showed lower phototoxicity compared to the widely used Fura-2. By analyzing Ca²⁺ signaling in NK cells during killing events, we observed that necrotic kills are associated with higher and shorter Ca²⁺ signals compared to apoptotic kills. Additionally, we investigated CAR-engineered NK cells, comparing their Ca²⁺ signaling profiles to those of primary NK cells. This combined analysis of Ca²⁺ signaling with automated cancer cell death assessment provides a robust framework for studying NK cell behavior, offering potential insights into their killing mechanisms. These findings could help optimize NK cell-based therapies by targeting Ca²⁺ signaling to enhance the efficiency of tumor cell killing.