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• Spring wheat was 15N-labeled via a split-root technique. • The NdfR was estimated by the isotope ratio and isotope mass balance approaches. • The isotope ratio approach underestimated NdfR before plant maturity. • The two approaches had no difference for estimation of NdfR after plant maturity. • Selecting appropriate calculation approach to quantify NdfR should be considered. Accurate quantification of nitrogen (N) derived from rhizodeposition (NdfR) is crucial to assess N stored in plants and the total N balance in plant-soil systems. However, there remains uncertainty in the estimates of NdfR for 15N-labeled plants with the split-root technique using the isotope ratio approach and the isotope mass balance approach. Here, we examined the two calculation approaches that estimate NdfR from 15N-urea (99 atom%) labeling of spring wheat (Triticum aestivum L.) using the split-root technique in a pot experiment over plant growth, and assessed the causative factors of their potential bias. The results showed that independent of the approach, the total amount of NdfR was 5.3–9.8 mg plant−1 and contributed up to 19.9–26.3% of total plant N throughout the growth period. At the heading and filling stages after labeling, the amounts of NdfR using the isotope ratio approach (5.3 and 6.6 mg plant−1, respectively) were significantly lower than those (7.1 and 7.8 mg plant−1, respectively) using the isotope mass balance approach, due to soil N losses and an uneven distribution of 15N. At the ripening stage after labeling, however, the differences in the amount of NdfR between the two approaches disappeared (9.8 and 9.6 mg plant−1, respectively). This indicated that the isotope ratio approach underestimates the amount of NdfR during the plant developmental period, while both approaches can be used to quantify the amount of NdfR at plant maturity. [ABSTRACT FROM AUTHOR]