Reliable and accurate monitoring systems for greenhouse gas emissions from animal feeding operations (AFOs) are crucial for establishment and enforcement of gas emission mitigation strategies. An automated multiplexing system for chamber-based monitoring of greenhouse and regulated gas emissions from manure sources was developed to examine spatial and temporal variability of emissions associated with manure management practices. The measurement system uses a Fourier Transformed Infrared (FTIR) spectroscopy analyzer for determination of up to 15 pre-programmed gas fluxes. Multiple chambers provide estimates of variance for emissions from different management practices. The objective of this paper is to demonstrate the robustness and reliability of the described system for monitoring gas emissions from AFOs. Evaluation of system performance was based on laboratory experiments using methane gas (CH4) to assess the accuracy of the chamber-based measurement system. We developed a method to generate constant emission of methane gas using a gradient-based technique for the reference gas flux. Three different emission rates were simulated. Statistical analysis, including ANOVA, was performed to determine the significance of gas flux estimates using the chamber-based estimate. A p-value ≤ 0.05 was considered to be statistically significant. The ANOVA tests indicated no statistically significant differences among estimated fluxes from each of the 12 evaluated chambers, with resulting p-values of 0.54, 0.58, and 0.80 for measurements of three different emission rates. In addition, the multi-chamber system measurements referenced to the gas fluxes estimated with the gradient-based method showed excellent accuracy with measurement biases less than 1%.