This paper reports the laboratory spectroscopic calibration of near- and mid- infrared tunable laser spectrometers used to determine in situ trace gases in the middle atmosphere of the Earth or in development for the investigation of the Martian atmosphere. The use of infrared absorption spectroscopy to measure gas concentrations requires a proper knowledge of the rotation–vibration spectra of the targeted molecules as well as a proper investigation of the tunable laser spectral emission properties. This last point is of particular importance for the use of new-generation lasers like quantum-cascade lasers or room-temperature multi-quantum wells laser diodes emitting between 2 and 3 μm. Purposely, we have developed various laboratory tunable laser set-ups to obtain accurate line strengths and pressure-broadening coefficients of atmospheric molecules and to test the performances of cutting-edge laser technology for trace gas sensing. In this paper, the spectroscopic calibration work is described. Several atmospheric applications of tunable laser are reported to stress the impact on concentration retrieval of a proper spectroscopic calibration work.