Direct measurement of ventilation rate in livestock housing can be a formidable task due to uncontrollable variations in fan and system performance as caused by factors such as operation static pressure, fan belt condition, and dust accumulation on shutters and blades. Indirect, CO2-balance method offers a potentially viable, more flexible alternative to estimating ventilation rate. The reliability of CO2 balance method depends on the validity of relationship between CO2 production and metabolic rate of the animals and the knowledge of CO2 generation by the housing environment. Metabolic rates of modern laying hens have recently been quantified in intensive large-scale laboratory measurements. However, performance of the indirect method remains to be evaluated under field production conditions. This paper compares ventilation rates of a commercial laying hen house with manure belt (manure removed daily) obtained from direct measurement based on in-situ fan performance and runtime vs. indirect determination based on CO2 balance. The results indicate that indirect determination based on CO2 balance was well in agreement with that of direct measurement. Application of the CO2-balance method to evaluate building ventilation rate can improve the affordability and versatility of poultry emission studies.