Quantification of root biomass through the conventional root excavation and washing method is inefficient. A pot experiment was conducted to estimate root-derived carbon (C) in soil. Spring wheat (Triticum aestivum L. cv. ‘Quantum’) was grown in plastic containers (6L) filled with sterilized sandy soil in a greenhouse. Plants were enriched with 13CO2 in a glass chamber twice at growth stages GS-37 and GS-59 for 70min at each time. In one treatment, roots were separated from soil at crop maturity, washed and dried for the determination of biomass. Isotope ratios were then separately analyzed for roots and soil. In a second treatment, roots were thoroughly mixed with the whole soil and representative samples were analyzed for 13C abundance at crop maturity. Control plants were untreated with 13C, in which roots were separated from soil. The root biomass was calculated based on the root-derived C, which was measured through 13C abundance in the soil and root mixed samples. A substantial amount of root-derived C (24%) was unaccounted while separating the roots from soil. Similarly, about 36% of the root biomass was underestimated if conventional root excavation and washing method is used. It has been shown that root biomass can be estimated more accurately from the root-derived C using 13C tracer method than the estimates made by the conventional excavation and washing method. We propose this as an alternative method for the estimation of root-derived C in soil, based on which root biomass can be estimated.