Glyphosate is one of the most widely used herbicides, and its human health effects remain debated. Risk assessment requires robust human biomonitoring (HBM) data generated using validated analytical methods. However, recent population studies employing commercial enzyme-linked immunosorbent assays (ELISA) have reported urinary glyphosate concentrations higher than those measured by mass spectrometry (MS), raising concerns about potential overestimation and data comparability. This study compares two MS/MS-based methods, including a novel ion chromatography-MS/MS approach, with two ELISA methods across external quality assessment (EQA) urine samples (n = 13), spiked urine samples (n = 3, 0.1–0.3 ng/mL), and real-world human urine samples (n = 40). The evaluated ELISA methods overestimated urinary glyphosate concentrations below 1 ng/mL in EQA and spiked samples and exhibited poor repeatability (often >20%) relative to MS/MS-based methods. Analyses of real-world samples confirmed this trend, with ELISA yielding higher quantification frequencies (70–100% vs 45–63%), higher median concentrations (1.06–1.10 vs 30%) than MS/MS methods. These findings indicate that the evaluated ELISA methods lack sufficient reliability for quantifying urinary glyphosate at concentrations relevant to nonoccupational exposure, where matrix-related interferences may compromise analytical performance. These results support the preferential use of MS/MS-based methods for generating robust HBM data in research and regulatory contexts.