Ventilation rate (VR) is one of the two key elements for quantifying aerial emissions from animal production facilities. Direct measurement of building VR can be challenging and impractical under certain circumstances, e.g., naturally ventilated animal housing. This study delineates VR of broiler houses with build-up litter as estimated via CO2 balance or building CO2 concentration. The indirectly derived VR compared favorably with the directly measured VR. Specifically, integration time of 30 min or longer leads to non-significant differences in VR between the indirect and the direct methods (P>0.2). Omission of CO2 generation by the litter from total house CO2 production results in an overall 7% underestimation of the building VR. The indirect method provides a possible, viable alternative for quantifying VR of naturally ventilated broiler confinement.