Levoglucosenone ((1S,5R)-6,8-dioxabicyclo[3.2.1]oct-2-en-4-one or LGO; CAS number: 37112-31-5) is an extensively studied biomass based chemical and its potential applications in organic synthesis, as well as in the extraction of various natural substances, have been established; therefore developing dependable, yet fast and simple analytical methods for determining LGO in the products of thermochemical biomass processing is of great importance. In this work different UHPLC methods have been developed and compared for the determination of LGO in wood pyrolysis products. In addition to UV detection, LGO determination conditions by single quadrupole mass spectrometry have been adjusted with and without chemical derivatization. The ionization of LGO in the positive and negative electrospray ionisation mode and the effect of a mobile phase additive on LGO and its degradation product ionisation have been evaluated. Using the UHPLC-UV system, a method for the quantification of levoglucosenone was developed and the analytical parameters of the method were determined. Utilising the developed UHPLC-UV method, studies of LGO stability in standard solution and in fast pyrolysis products were carried out in various solvent systems at different temperatures and pH levels. The main LGO primary degradation product has been confirmed to be furfural. It has been concluded that the degradation rate of LGO is significantly affected by the solvent and storage temperature, so it is essential that LGO standard solutions and samples are analysed when freshly prepared. Using a UHPLC-MS system and derivatization with 2,4-dinitrophenylhydrazine (DNPH), a method for the determination of LGO was developed and the method analytical parameters were tested. To sum up, quantitative determination of LGO in pyrolysis products can be performed by reversed phase UHPLC-UV without any particular sample preparation, but in the case of UHPLC-MS it is necessary to derivatize LGO with DNPH to ensure ionisation of LGO. The additional work, requiring MS detection, can be outweighed by the greater selectivity compared to that of the more straightforward UV detection method in cases of incomplete chromatographic separation.