Abstract One of the major constraints of measurements of atomic hydrogen densities using two-photon absorption laser induced fluorescence in most plasma and combustion environments is the determination of fluorescence decay times ( τ fluo H ), especially when using nanosecond-lasers or slow acquisition systems. Therefore, it is necessary to identify the depopulation processes of the laser excited level in order to correctly estimate τ fluo H . In this study, depopulation mechanisms of atomic hydrogen excited by two-photon absorption to the n = 3 level (H( n = 3)) have been investigated using a picosecond-laser excitation and acquisition of fluorescence by a streak camera, which allowed for direct measurement of τ fluo H and hence, the atomic hydrogen densities, in an H 2 microwave plasma operating in the pressure range 20–300 Pa. By combining these measurements with a detailed H( n = 3) collisional radiative depopulation model, it was found that full mixing amongst the H( n = 3) sub-levels occurs in our discharge conditions, even at a pressure as low as 20 Pa. Moreover, it is also seen that the Lyman β line is only partially trapped, as its escape factor Λ 31 decreases from 0.94–0.98 down to 0.58–0.86 while the measured atomic hydrogen density rises from 8 ± 5 × 10 19 m − 3 to 9 ± 6 × 10 20 m − 3 . This means that the radiative decay rate of H( n = 3) atoms varies with pressure and the classical Stern–Volmer method used to determine the quenching cross-section of excited H( n = 3) in collisions with H 2 molecules, σ Q H n = 3 / H 2 , is not valid for our measurements. We used two different physics-based approaches, and show that the quenching cross-section σ Q H n = 3 / H 2 lies in the range 90– 106 × 10 − 20 m 2 , which can be averaged as 98 ± 8 × 10 − 20 m 2 . This substantially improved estimation of σ Q H n = 3 / H 2 obtained in this work will be useful for the accurate estimation of H( n = 3) fluorescence decay times and therefore the atomic hydrogen densities.