The physiological and pathophysiological importance of intracellular redox active "labile" iron has created a significant need for improved noninvasive diagnostic tools to reliably monitor iron metabolism in living cells. In this context, fluorescent iron-sensitive chemosensors in combination with digital fluorescence spectroscopic methods have proven to be highly sensitive and indispensable tools to determine cellular iron homeostasis. Recently, application of fluorescent iron sensors has led to the identification of a complex sub-cellular iron compartmentation. Cell organelle-specific iron sensors will significantly contribute to enhance fundamental knowledge of cellular iron trafficking, representing a crucial prerequisite for the future development of therapeutic strategies in iron dysregulatory diseases. Here we present physicochemical characterization and functional investigation of a new 3-hydroxypyridin-4-one based fluorescent iron(III) sensor, exclusively monitoring labile iron pools in the endosomal/lysosomal compartments. In vitro studies of the fluorescein labeled probe were carried out in murine bone marrow derived macrophages. Endosomal/lysosomal accumulation of the probe was revealed by confocal microscopy. Flow cytometry analyses demonstrated high sensitivity of the probe towards exogenous alterations of intracellular iron concentrations as well as in response to the chelation potency of iron chelators, clinically approved for treatment of iron-overload related diseases.