Due to its richness in phenolic compounds and other bioactive substances, honey is well-known for its antioxidant ability and since it has beneficial effect od health, it is used in folk medicine. Antioxidant activity is determined with in vitro assays, using spectrophotometrics methods. However, the sample preparation can lead to different results of the analysis. Thus, in this bachelor thesis, we aim to research the effect of the sample preparation on honey antioxidant activity determination. Our aim was to determine total phenolic and total flavonoid content in the honey samples and their antioxidant activity. The samples that we analysed were of different botanical origin, and of different of colours, prepaired on three different ways. The analyses were carried on the samples od acacia, floral and forest types of honey. We measured their water content (refractometric assay), electric conductivity (conductometric assay) and colour (with chromameter). For the spectrophotometric assays we prepaired the honey samples in three different ways: by dissolving honey in 1) water, 2) 70% ethanol or 3) acidified water, then extracted on solid phase (SPE) and eluted phenolic fractions in methanol/acetonitrile solution. The results revealed positive correlation between phenolics content and antioxidative ability. The values of these parameters are linked to the botanical origin of honey, its colour and its electric contuctivity. The analyses od the total phenolic content, using Folin-Ciocalteu reagent, of the total flavonoids content using aluminium chloride reagent as well as the analysis of the antioxidant activity using the DPPH reagent, revealed the effect of the sample preparation on measured values. Water and ethanol extracts resulted in higher values than the samples, extracted on SPE. This is due to the presence of sugar and other reducing compounds in honey, which hamper analysis. Thus, we discovered that the samples, that were prepaired by using the solid-phase extraction method produce the most reliable results, especially when the determination of the total flavonoid content is concerned.