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The phosphonate group is a key pharmacophore in many antiviral, antimicrobial, and antineoplastic drugs. Due to its high polarity and short retention time, detecting and quantifying such phosphonate-containing drugs with LC/MS-based methods are challenging and require derivatization with hazardous reagents. Given the emerging importance of phosphonate-containing drugs, developing a practical, accessible, and safe method for their quantitation in pharmacokinetics (PK) studies is desirable. NMR-based methods are often employed in drug discovery but are seldom used for compound quantitation in PK studies. Here, we show that proton–phosphorous (1H–31P) heteronuclear single quantum correlation (HSQC) NMR allows for the quantitation of the phosphonate-containing enolase inhibitor HEX in plasma and tissues at micromolar concentrations. Although mice were shown to rapidly clear HEX from circulation (over 95% in <1 h), the plasma half-life of HEX was more than 1 h in rats and nonhuman primates. This slower clearance rate affords a significantly higher exposure of HEX in rat models compared to that in mouse models while maintaining a favorable safety profile. Similar results were observed for the phosphonate-containing antibiotic, fosfomycin. Our study demonstrates the applicability of the 1H–31P HSQC method to quantify phosphonate-containing drugs in complex biological samples and illustrates an important limitation of mice as preclinical model species for phosphonate-containing drugs.