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Environment-sensitive probes constitute powerful tools for monitoring changes in the physico-chemical properties of cell plasma membranes. Among these probes, 3-hydroxyflavone probes are of great interest due to their dual emission and ratiometric response. Here, three probes derived from the parent F2N12S were designed, characterized and applied to monitor the membrane changes occurring during apoptosis. These three probes were designed to orient the dye vertically in the membrane. They differ by the length of their alkyl chains (from 4 to 8 carbons), which were included to optimize their affinity to the lipid membranes. Among these three probes, the one with medium chain length (hexyl) showed the best affinity to model and cell membranes, while the one with the longest alkyl chains (octyl) did not efficiently stain the membranes, probably due to aggregation. The new probes were found to be more sensitive than F2N12S to both the lipid phase and surface charge in lipid vesicles and to loss of lipid order in cell plasma membranes after cholesterol extraction. The one with the shortest (butyl) chains was found to be the most sensitive to apoptosis, while the one with medium-length (hexyl) chains was the brightest. Interestingly, apoptosis induced by different agents led to similar spectroscopic effects to those produced by the loss of lipid order and change in the surface charge, confirming that apoptosis decreases the lipid order and increases the negative surface charge in the outer leaflet of cell membranes. In conclusion, these studies report the relationship between the probe structures and their sensitivity to lipid order, surface charge and apoptosis and propose new probes for membrane research.