The concentration of lactose, the primary carbohydrate in human milk, changes dynamically throughout lactation, making its accurate monitoring essential. However, carbohydrate quantification using mid-infrared (MIR) breast milk analyzers currently employed in clinical settings cannot distinguish between lactose and human milk oligosaccharides (HMOs). Against this backdrop, we developed a method for lactose quantification in human milk using recently developed benchtop nuclear magnetic resonance (NMR) instruments. By optimizing the solvent suppression conditions and selecting an appropriate lactose-derived NMR signal region for quantification, we demonstrated that quantification comparable to that obtained with an 800 MHz high-field NMR instrument is achievable even with a 60 MHz benchtop system. Furthermore, by developing a correction method to account for HMO interference arising from the spectral overlap with lactose signals, even in low resolution benchtop NMR spectra, further improvements were achieved in quantification precision. After correction, the agreement between the 60 and 800 MHz results was supported by Deming regression (slope 0.994) and Bland-Altman analysis (mean bias -0.430 mM). Interference from HMOs was particularly pronounced in colostrum and exhibited substantial inter-individual variation. These findings highlight the potential of benchtop NMR-based methods for lactose quantification in human milk and support further evaluation in broader research and clinical settings.