Human-induced pluripotent stem cells (hiPSCs) have broad applications in basic research and cell-based regenerative medicine. Maintaining a stable genome in culture is crucial for the clinical use of hiPSCs. Indeed, their continuous propagation drives genetic instability, particularly copy number variants (CNV). Among the different methods available to identify gene alterations, digital PCR (dPCR) is a precise, rapid, and cost-effective tool for the sensitive and absolute quantification of CNV. Unlike conventional methods limited to two targets, multiplex dPCR assays allow the simultaneous detection of four targets. In this method paper, we describe strategies for multiplex dPCR assays to detect major CNVs using only small amounts of DNA extracted from hiPSCs. We confirmed this approach applicability for hiPSCs through validation following the International Council for Harmonization (ICH) guidelines that cover different aspects, such as specificity, precision, robustness, and limit of detection. The simultaneous measurement of four targets was possible through the generation of distinct fluorescence endpoints for each target by adjusting the concentrations of primers and/or probes. Compared to the classical dPCR (the “gold standard”), these multiplexed dPCR assays displayed 100% specificity and sensitivity for the simultaneous detection of CNVs. This study highlights the key role of multiplexing for monitoring genetic alterations in hiPSCs and for setting the standards required for the process safety evaluation of hiPSC-based stem cell therapies.