Quantitative nuclear magnetic resonance (qNMR) has emerged as an easy, rapid and reproducible method for various pharmaceuticals. In the current study, a general qNMR approach for calibrating the purity of the thiopeptcin reference standard (also known as nocathiacin I) was developed using sulfadoxine as an internal standard. Experimental conditions, such as the relaxation delay time and number of scans, were systematically optimized, and the method was validated with different analytical parameters, including selectivity, stability, linearity, precision and robustness. To examine the reliability and feasibility of the present qNMR method, there was no significant difference in the quantification of this complex cyclic peptide compared to the mass balance method. The present study further exemplified that qNMR is a reliable and valuable approach for the assessing of absolute purity of small-molecule pharmaceuticals, which provides a useful tool for drug discovery and development.