A comprehensive profiling of cardiotoxicity early in drug discovery and development can aid in reducing late-stage attrition and establishing risk-mitigation strategies during clinical development. In most cases, multiple assay platforms and instrument-specified plate formats are required for this type of approach. In this study, we evaluated both functional and structural endpoints associated with cardiotoxicity in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) cultured in a single 384-well plate. We measured intracellular Ca2+ transit, caspase 3/7 activation and plasma membrane permeabilization sequentially in the same plate via a series of assay readouts. A set of cardiac ion channel modulators (dofetilide, sotalol, nifedipine and mexiletine) and chemotherapeutics (tamoxifen, nilotinib, sunitinib and doxorubicin) was tested at clinically relevant concentrations for effects on intracellular Ca 2+ transits after a short-term (30 minutes) exposure, and plasma membrane permeabilization and caspase 3/7 activation after a long-term (72 hours) exposure. Intracellular Ca2+ transits were monitored by fluorescent images taken with a high-speed camera in beating cardiomyocytes loaded with Cal520® Ca2+ dye, permeabilized plasma membrane (for dead-cell detection) was identified with live-stain DRAQ7TM nuclear dye and activation of caspase 3/7 was determined biochemically with the Caspase-Glo® 3/7 Assay kit. Multiple endpoints derived from Ca 2+ transits, including beat rate, calcium transit duration (CTD) measured at 30% or 90% from peak and corrected by inter-peak interval (IPI), along with CTD triangulation, beat rhythm, short- or long-term variability of CTD90 and IPI Poincaré plots, were used to assess drug effects on intracellular Ca2+ cycling and arrhythmogenicity. Increases in positive nuclear staining for DRAQ7TM and caspase 3/7 activity represented structural cardiotoxicity. We found that increased CTD triangulation, development of arrhythmic events and both the short- and long-term variability of CTD90 or IPI were robust indicators of functional effects. Positive nuclear staining for DRAQ7TM was a robust indicator of structural effects. Accordingly, dofetilide and sotalol were identified as primarily arrhythmogenic, doxorubicin was primarily structurally toxic, while nilotinib and sunitinib were both arrhythmogenic and structurally toxic. The use of these endpoints in a single plate format simplifies the cardiotoxicity assessment and does not require multiple cell plates for measurements.