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DOSY NMR spectroscopy was used for the determination of molecular weight of heparin and LMWH.PLS model using GPC reference data was developed and completely validated.The method was applicable to heparin and LMWH of different animal origin.The method can be used as a fast complementary measurement to standard NMR assay.Apart from the characterization of impurities, the full characterization of heparin and low molecular weight heparin (LMWH) also requires the determination of average molecular weight, which is closely related to the pharmaceutical properties of anticoagulant drugs.To determine average molecular weight of these animal-derived polymer products, partial least squares regression (PLS) was utilized for modelling of diffused-ordered spectroscopy NMR data (DOSY) of a representative set of heparin (n = 32) and LMWH (n = 30) samples. The same sets of samples were measured by gel permeation chromatography (GPC) to obtain reference data. The application of PLS to the data led to calibration models with root mean square error of prediction of 498 Da and 179 Da for heparin and LMWH, respectively. The average coefficients of variation (CVs) did not exceed 2.1% excluding sample preparation (by successive measuring one solution, n = 5) and 2.5% including sample preparation (by preparing and analyzing separate samples, n = 5). An advantage of the method is that the sample after standard 1D NMR characterization can be used for the molecular weight determination without further manipulation. The accuracy of multivariate models is better than the previous results for other matrices employing internal standards. Therefore, DOSY experiment is recommended to be employed for the calculation of molecular weight of heparin products as a complementary measurement to standard 1D NMR quality control. The method can be easily transferred to other matrices as well.