Ammonia (NH3) emission from animal feeding operations (AFOs) has caused concerns on public health and environmental degradation, such as ecosystem acidification, eutrophication, and formation of PM2.5 fine particles. Current ammonia emission measurement methodologies are accurate and reliable, but time consuming, expensive, and impractical for most facilities. In the present study, an alternative and cost effective mass balance methodology was developed to predict the ammonia emission from animal facilities. The mass balance equations have been developed to eliminate needs for tracking manure flow rate to obtain accurate NH3 estimation. The methodology was applied to three manure-belt layer poultry houses with approximately 150,000 birds in each house in Ohio and validated using continuous ammonia emission measurement data. Feed, manure and egg samples were collected from the three houses in three seasons (cold, mild, and hot) to evaluate the seasonal variation of ammonia emission from the poultry facilities. Results show that this alternative mass balance method can estimate NH3 emission from manure-belt poultry layer house effectively. NH3 emission rate from manure belt poultry layer houses with manure removal every 3.5 to 5 days was 0.07-0.37 g NH3 bird-1day-1. These results agrees well with the NH3 emission values published in the previous literatures (0.027-0.616 g NH3 bird-1day-1), but were lower than the NH3 emission rate (0.1-0.86 g NH3 bird-1day-1) measured using continuous monitoring system. In the comparison analysis of NH3 measurement and estimation emissions, Normalized Mean Error (NME), Normalized Mean Square Error (NMSE) and Fractional Bias (FB) are calculated to be 52.05%, 85.32% and -70.36% respectively. This study suggests that manure removal time interval and air temperature can be important factors impacting NH3 emission. This mass balance method can only estimate total nitrogen loss in a whole production process, which is an upper bound of NH3-N loss. It is needed to quantify other nitrogen compound gas emissions, such as N2O, NOx, N2 for accurate NH3 emission estimation.