Loss of lysosomal membrane integrity results in leakage of lysosomal hydrolases to the cytosol which might harm cell function and induce cell death. Destabilization of lysosomes often precede apoptotic or necrotic cell death and occur during both physiological and pathological conditions. The weak base acridine orange readily enters cells and accumulates in the acidic environment of lysosomes. Vital staining with acridine orange is a well-proven technique to observe lysosomal destabilization using fluorescence microscopy and flow cytometry. These analyses are, however, time consuming and only adapted for discrete time points, which make them unsuitable for large-scale approaches. Therefore, we have developed a time-saving, high-throughput microplate reader-based method to follow destabilization of the lysosomal membrane in real-time using acridine orange. This protocol can easily be adopted for patient samples since the number of cells per sample is low and the time for analysis is short.