As the amount of fossil fuels is limited and the fuel itself is harmful to the environment, it is recommended to switch to renewable energy sources, including biodiesel. Biodiesel is an increasingly popular fuel today due to its biodegradability, low emissions, less environmental pollution and non-toxicity. Most often, the production of biodiesel takes place by the process of transesterification of various oils and fats with methanol in batch reactors, with the addition of acids, bases or enzymes as catalysts. The goal of production is to get the primary product as pure as possible, which can be used immediately as a fuel with small amount ofby-products as possible in order to avoid often very expensive purification processes. To make production process as successful as possible, it is necessary to continuously monitor and analyse the reaction products during the process. Although there are already various methods of analysis of biodiesel and glycerol, new methods are being researched every day, the main goal of which is to be as simple, precise and fast as possible. In this work biodiesel was synthesized by enzymatically catalysed transesterification using sunflower oil and methanol as substrates. During the process, the concentration of glycerol, as one of the indicators of the success of the process, was monitored. At the very beginning, an analysis of the fatty acid esters concentration using gas chromatography was performed from which the theoretical value of the glycerol concentration was calculated. The calculated glycerol concentration was compared with the glycerol concentration obtained by analysis using standard methods on a UV-VIS spectrophotometer and by the gas chromatography. Because neither of the methods gave satisfactory results, meaning both analyses showed significant deviations from the theoretical value of glycerol, the analysis on NIR spectrometer was proposed. During the synthesis reaction, the analysis of biodiesel concentration was performed in such a way that samples were taken at certain time intervals for the off-line analysis and on-line in such a way that the concentration was determined continuously by screening the reaction mixture which was passing through a flow cell of the NIR spectrometer. The NIR spectroscopy method proved to be the best, with the greatest agreement of the measured glycerol concentration with the theoretically calculated concentration.