Hydrogen atoms are key species in combustion of hydrogen/hydrocarbon fuels. Interference-free detection of hydrogen atoms natively generated in flames using femto-second laser-induced fluorescence (LIE) was investigated employing two colors, i.e., 243 nm and 486 nm, as excitation source: two-photon excitation followed by a relay one-photon excitation. This strategy was compared with another commonly adopted two-photon LIF strategy using 205 nm for excitation. The potential interferences were investigated, and a direct verification method was proposed to prove this strategy be interference-free, and imaging of hydrogen atoms natively generated in methane/air flames was achieved. (C) 2016 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.