Vitamin-fortified fruit juices may be a convenient source of biologically active compounds including the folic acid and folates, its derivatives, which naturally occur in fruits. However, those compounds show a low chemical stability, which makes the determination of their content very difficult. Based on the reference literature and the authors’ own study, it has been found that any biological material needs different conditions to be applied to the effective extraction and hydrolysis of its components prior to their determination using a HPLC method. The objective of the research was to determine the effect of different conditions of extraction (pH of buffer, time/temperature) and hydrolysis (RP – quantity of plasma in the blood of rats as a source of folate conjugase) on the content of folic acid and folates in the material analyzed. The highest content of folic acid (32.62 – 34.53 µg/ 100 ml juice) was determined when the extraction was performed using a phosphate buffer of pH = 7.0 at 100º C for 15 minutes. The highest content of methyl form (4.23 – 4.29 µg 5CH₃FH₄/ 100ml juice) was determined in the case of the extraction carried out in a phosphate buffer of pH=6.1 at 100º C for 15 minutes. Different amounts of plasma in the blood of rats added under the same conditions of extraction had not any significant effect (α =0.05) on the determined content values of both the folic acid and 5CH₃FH₄.