In this doctoral thesis, the possibilities of applying the LC-SPE/cryo NMR method for monitoring chemical reactions and determining the structure of by-products and/or degradation products on-line, with an emphasis on bioactive molecules and drugs, were explored. Three different model systems were explored for methodology development. For each model system, the parameters for chromatographic separation of the reaction mixtures components, as well as parameters for the efficient extraction of the selected analytes on SPE-sorbents, were optimized. Hence, the influence of the SPE-parameters on the NMR cryoprobe sensitivity was also evaluated. NMR spectroscopy and LC-MS were used for identification and structural characterization of the extracted mixture components. The first model system were carbohydrazides and their derivatives, which are known as chelating reagents and also show antioxidative and antiepileptic effects. Sensitivity of LC-SPE/cryo NMR system was determined by analyzing carbohydrazides. Etodolac, a nonsteroidal anti-inflammatory drug, was chosen as the second model system. During the analysis of 7-ethyltryptophol, a starting material in the synthesis of etodolac, 17 impurities were identified and structurally characterized. The third model system were macrozones, new amide conjugates of azitromycin and thiosemicarbazide derivatives which exhibit good antibacterial activity against susceptible and resistant bacterial strains. Six reaction mixtures from three different classes of newly synthesized macrozones were analysed. Identified impurities were classified as macrolide and non-macrolide related by-products and degradation products, as well as macrozone diastereomers. Configurations of diastereomers were proposed on the basis of NOESY NMR spectroscopy data and molecular dynamic simulations. This research resulted in development of a reliable, faster and more efficient methodology for complex mixture analysis using significantly smaller amount of sample and organic solvents in comparison with standard off-line methods. LC-SPE/cryo NMR method can be applied for the extraction, purification and characterization of newly synthesized compounds and/or impurities from reaction mixtures.