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Abstract The Pico-STRAT Bi Gaz spectrometer provides in situ mixing ratio measurements of water (H₂O) and methane (CH₄) [or carbon dioxide (CO₂)] under balloon. The instrument was flown in the tropical upper troposphere and lower stratosphere in 2019/20 and 2021/22 during the Strateole 2 campaigns for a total of five flights of 20–80 days between 18- and 20-km altitude. In this frame, in situ measurements of water vapor and methane were performed every 4–12 min in the equatorial tropopause layer. On several occasions, water vapor measurements of Pico-STRAT Bi Gaz have been compared with localized measurements from the Fluorescence Lyman-Alpha Stratospheric Hygrometer for Balloon (FLASH-B) Lyman-αhygrometer and vertical profiles of the NOAA Global Monitoring Laboratory (GML) frost point hygrometer over Hilo, Hawaii. Pico-STRAT Bi Gaz measurements agreed with the FLASH-B hygrometer to within 2.2% ± 5.3% between 18.2 and 18.7 km in 2021 and to within 1.3% ± 5.3% near 19 km in December 2019. Pico-STRAT Bi Gaz agreed with NOAA’s frost point hygrometer (FPH) hygrometer to within 1.2% ± 4.1% between 18 and 19 km on four occasions during the two campaigns. These are within both instruments’ uncertainties. Methane measurements from Pico-STRAT Bi Gaz have been compared with in situ measurements from the whole air sampler (WAS) instrument, flown aboard the NASA WB-57 aircraft during the Asian Summer Monsoon Chemical and Climate Impact Project (ACCLIP) 2022 campaign over South Korea, 8 months after the Pico-STRAT Bi Gaz overpass. The relative difference between both instruments is found to be −0.1% ± 0.9% within the altitude range from 17 to 19 km and within the Pico-STRAT measurement uncertainty.