The objective of this study was to examine the accuracy of urinary creatinine excretion as a predictor for urinary nitrogen (UN) output in dairy cows fed adequate and protein deficient diets. To determine dietary protein requirements and measure efficiency of N use in lactating dairy cows, N balance must be calculated. This requires measurement of N excreted in milk, feces and urine. Performing total urine collection via catheter to measure UN output is labor intensive and puts cows at risk for infection. As an alternative method, creatinine excretion is commonly used as a predictor of daily urine output. However, the accuracy of this method has been questioned, and previous research has not examined creatinine’s accuracy in cows fed adequate vs protein deficient diets. This study used 21 mid-lactation Holstein cows in 2 blocks. For 4 weeks, half the cows consumed protein-adequate diets and the other half consumed protein-deficient diets, then all cows were fitted with urinary catheters connected to total collection cans containing 50% sulfuric acid for 72h. Every 9 h, urine in the can was removed and measured, acid was re-added, and urine samples were taken from the cows and collection cans. Cows were milked 2× daily, and BW was taken 3× in the week before total collection. Urine samples for each cow from the collection can and cow were composited, and creatinine and N content were measured. Daily total creatinine output was estimated as 29 mg/kg BW. Data were analyzed by t-test and correlation. In both experiments, creatinine concentration in samples taken from cows accurately represented samples from cans. However, creatinine-estimated and actual daily urine output values were significantly different (P < 0.05), even though the correlations between them were moderate (0.51 in experiment 1 and 0.94 in experiment 2). When N output was calculated using creatinine-estimated vs actual urine output values, creatinine underestimated actual N output by 17% on average. Using creatinine to estimate urine volume can be used to see relative differences among cows but values are not always quantitative and should be used with caution.