This paper presents a general approach to quantify the absorption model uncertainty due to uncertainty in underlying spectroscopic parameters. The approach is applied to radiative transfer calculations in the 20–60 GHz range, which is commonly exploited for atmospheric sounding by microwave radiometer (MWR). The approach however is not limited to any frequency range, observing geometry, or particular instrument. In the considered frequency range, relevant uncertainties come from water vapor and oxygen spectroscopic parameters. The uncertainty of the following parameters is found to dominate: (for water vapor) self and foreign continuum absorption coefficients, line broadening by dry air, line intensity, temperature-dependence exponent for foreign continuum absorption, and line shift-to-broadening ratio; (for oxygen) line intensity, line broadening by dry air, line mixing, temperature-dependence exponent for broadening, zero-frequency line broadening in air, temperature-dependence coefficient for line mixing. The full uncertainty covariance matrix is then computed for the set of spectroscopic parameters with significant impact. The impact of the spectroscopic parameter uncertainty covariance matrix on simulated downwelling microwave brightness temperatures (TB) in the 20–60 GHz range is calculated for six atmospheric climatology conditions. The uncertainty contribution to simulated TB ranges from 0.30 K (sub-Arctic winter) to 0.92 K (tropical) at 22.2 GHz, and from 2.73 K (tropical) to 3.31 K (sub-Arctic winter) at 52.28 GHz. The uncertainty contribution is nearly zero at 55–60 GHz frequencies. Finally, the impact of spectroscopic parameter uncertainty on ground-based MWR retrievals of temperature and humidity profiles is discussed.