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Zinc pyrithione is ubiquitous in commercial products particularly antidandruff shampoos. For the efficacy of zinc pyrithione therapeutic cleansers to be assessed accurately, the distribution of particles on the scalp needs to be visualized. Currently, no technique is available which provides the chemical specificity and sensitivity required. Here, we report application of fluorescence‐lifetime imaging microscopy (FLIM) for high‐contrast mapping of zinc pyrithione distribution on the scalp. Characterization of the zinc pyrithione emission by using both one‐photon excitation at five specific wavelengths and two‐photon excitation in the range of 740–820 nm revealed its FLIM fingerprint—a characteristic short average time‐weighted emission lifetime of ΤZnPT = 250 ps. Bandpass‐filtering FLIM signals at ΤZnPT enabled an efficient discrimination between the zinc pyrithione and major endogenous skin species in comparison with that of the conventional reflectance confocal microscopy. Our findings provide means for in vivo high‐sensitivity assaying and high‐contrast imaging of zinc pyrithione in biological systems.

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