The main objective of this study was to establish a protocol to validate urine spot samples to estimate N excretion and microbial synthesis in goat and sheep; and to study factors that affect daily creatinine and purine derivatives (PD) urinary excretion. Also a performance trial was carried out to compare goat and sheep slaughtered after different feedlot periods. Twelve Boer goats (20.6 kg ± 3.4 initial BW) and 12 Dorper sheep (18.4 kg ± 2.3 initial BW), all 4-mo-old, males, were used. Eight animals (4 goats and 4 sheep) were randomly allocated to be slaughtered at 28, 56, and 84 d in feedlot. The experiment was conducted in a completely randomized design in a 2 × 3 factorial scheme, in which the factors were both species and the 3 feedlot periods. Diet consisted of 50% sorghum silage and 50% concentrate on a DM basis. Nutrient intake was higher (P < 0.01) for sheep than goats. Apparent digestibility of nutrients was similar (P > 0.05) in both species. Sheep had greater (P < 0.01) ADG and final BW than goats. Fat deposition and fat:muscle ratio was higher (P < 0.01) in sheep carcasses. Sheep had higher N urinary (P = 0.02) excretion and N retention (g/d; P < 0.01) than goats. Urinary N excretion increased linearly (P < 0.01) in response to feedlot period. However, feedlot did not affect (P = 0.20) N retention, but linearly reduced the relationship between N retained and ingested (P = 0.04) or apparently digested (P < 0.01). Microbial efficiency (P > 0.05) did not differ between species. Creatinine excretion (C mg/d; P < 0.01) was higher in sheep than goats. Purine derivatives (Ŷ) were related closely with OM intake (Ŷ = 0.013±0.0007X; r2 = 94). A difference (P < 0.01) was found between the allometric model for creatinine excretion (Ŷ) and muscle weight (X) for both species, and the following equations were obtained: Ŷ = 89.04(±31.44)X0.9797(±0.16) for goats and Ŷ = 109.8(±47.50)X0.8002(±0.20) for sheep. Creatinine concentration was greater during nocturnal than diurnal periods, with lower diurnal fluctuations. Sampling time did not affect (P = 0.27) the PD:C ratio. The urea (U):C ratio was higher (P < 0.01) in sheep than goats, and was also higher (P < 0.01) during diurnal than nocturnal sampling periods. Our results suggest that it is necessary to take 2 and 3 spot urine samples after feeding to estimate N compounds excretions in goats and sheep, respectively.