Ammonia (NH₃) emissions from poultry animal feeding operations (AFOs) have caused health and environmental concerns. Current NH₃ emission measurement methods are accurate and reliable but also time-consuming, expensive, and impractical for most animal facilities. In this study, an alternative mass balance method was developed to effectively predict NH₃ emissions from manure belt (MB) poultry layer facilities. This method can eliminate the need for tracking manure flow rates in traditional mass balance analyses for estimation of ammonia nitrogen (NH₃-N) emissions. It was applied to three MB layer poultry houses in Ohio, with approximately 160,000 hens in each house, and validated using continuous NH₃ emission measurement data. Feed, manure, and egg samples were collected from the three houses in different months over a year to evaluate possible seasonal variation in NH₃ emissions from the poultry houses. The estimated NH₃-N emissions from houses 1, 2, and 3 were 0.394 ±0.143, 0.293 ±0.1, and 0.284 ±0.129 g NH₃-N hen^-1 d^-1, respectively, and the measured NH₃-N emission rates were 0.200 ±0.067, 0.220 ±0.036, and 0.237 ±0.211 g NH₃-N hen^-1 d^-1, respectively. These results are comparable with NH₃-N emission rates published in the literature (0.024 to 0.592 g NH₃-N hen^-1 d^-1). A statistical comparison of the measured and estimated NH₃-N emissions showed that the root mean square error (RMSE), normalized mean square error (NMSE), and fractional bias (FB) were 0.179 g NH₃-N hen^-1 d^-1, 0.426, and 0.457, respectively. These statistical parameters indicated that the estimations were acceptable according to the criteria of NMSE < 0.5 and FB < 0.5. The results showed that this alternative mass balance method could be used to estimate NH₃-N emissions from MB poultry layer houses. However, the method estimates total nitrogen gas emissions, which is an upper limit of NH₃-N emissions. A minimum of 22 sampling and modeling events is suggested for reliable estimation of NH₃-N emission factors for MB poultry layer houses using the alternative mass balance method with a 90% confidence level (α = 0.1) and a maximum error of 15%.