Heme is an essential prosthetic molecule for life activities and is well known to act as the active center of many proteins, however, labile heme (LH) released from proteins is a harmful molecule that produces reactive oxygen species and must be strictly controlled. Recently, LH has been suggested to function as an important molecule for diverse physiological responses. Quantitative analysis of the intracellular dynamics of LH is essential for understanding its physiological functions, a substantially practical method has not been established. Here, we successfully developed an alternative method that can be used to complement quantification of the dynamics of intracellular LH using H-FluNox, an activity-based specific fluorescent probe recently constructed. Our newly established method should be effective in elucidating the physiological functions of LH.