Anthracycline antibiotics are a class of drugs used in cancer chemotherapy. Unfortunately, they are characterized by serious side effects, particularly life-threatening cardiotoxicity. For this reason, many scientists are looking for new anthracycline derivatives, which would have similar efficacy but reduced toxicity. The biological activity of anthracycline antibiotics, their uptake by cancer cells and transport in the organism are dependent on physicochemical properties, such as, pKa and logP. In addition to physicochemical properties, also pharmacokinetic profile is extremely important for the future of the compound, therefore, the key point to selecting potential drug candidates is to determine, at the initial stage of testing, their physicochemical properties and pharmacokinetic profile. During the conducted studies new bioanalytical methods using capillary zone electrophoresis and microemulsion elecrokinetic capillary chromatography were developed. First method allowed to determine pKa values, whereas second were used to determine logDpH10 of tested compounds. In the next step new methods using ultra-efficient liquid chromatography in combination with tandem mass spectrometry were created and validated. These methods were used to determine the concentration of new anthracycline antibiotics in the mice plasma in order to assess the pharmacokinetic profiles of new compounds after cassette dosing.