Previous studies over two hepatoselective NO-donors revealed that only one of them, V-PYRRO/NO attenuated liver steatosis in mice, while its analog V-PROLI/NO was not effective. These studies suggest, that the explanation of distinct pharmacological effectiveness may be in different pharmacokinetic and metabolic profiles of studied compounds. Therefore, the purpose of this study was to develop and validate methods for evaluating the pharmacokinetic profile and metabolism of the V-PYRRO/NO and V-PROLI/NO. In the course of the work the method for determination of studied compounds in biological matrices, the method for evaluating the activity of cytochrome P450, and an alternative method of examining the metabolism using electrochemical oxidation were prepared. In the second part results indicated that indeed V-PYRRO/NO and V-PROLI/NO were characterized by a different pharmacokinetic profile. V-PYRRO / NO was metabolized in the liver by the cytochrome P450, which correlates with its pharmacological effect, while V-PROLI/NO was mainly excreted by the kidneys unchanged. Nonalcoholic fatty liver disease, has significantly amended the pharmacokinetic profile of both compounds, suggesting that the liver diseases can affect the pharmacokinetic profile of drugs metabolized in the liver, but also drugs excreted by other routes.