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The aim of this study was to validate a mass balance method based on N and ash in manure to estimate NH3-N losses. Six multiparous Holstein cows were used in a replicated 3 x 3 Latin square. Three diets, corn silage (CS), alfalfa silage (AS) and corn silage + urea (CSU) with different N:Ash ratios, 0.41, 0.37, 0.47 respectively, were fed. During total manure collection, urine and feces samples were used to prepare a slurry (1200 g) in the same proportion as excreted by each cow. Slurries were incubated for 3 d. Initial and final N concentrations and slurry masses were used to determine N losses. Nitrogen loss was estimated as: (N Intake – N Milk) – [N/Ash*(Ash Intake – Ash Milk)]. Average manure output was 65.4 kg/d with cows fed AS and CSU excreting 18.6 kg/d of urine followed by CS at 14.5 kg/d (P= 0.02). Manure N excretion was highest for cows fed CSU followed by AS and CS (422, 392 and 343 g/d, P<0.01). Ash excretion in manure was highest (P<0.01) for cows fed AS (1134 g/d) followed by CS and CSU (936 g/d). Measured NH3-N loss was 30, 41, and 70 g/3 d for AS, CS and CSU and calculated losses were 17, 31, and 75 g/3 d, respectively. Average estimated NH3-N losses did not differ from measured losses (P= 0.15); however, method underestimated NH3-N losses in cows with negative N balance and overestimated it for cows in positive N balance.