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Doxorubicin (DOX) belongs to the group of anthracycline antibiotics with very effective anticancer properties. On the other hand, the cardiotoxic effects limit its application over the maximum cumulative dose. To overcome this obstacle, encapsulation of this drug into the protective nanotransporter such as apoferritin is beneficial. In this study, fluorescent behavior of DOX in various solvents was determined by fluorescence spectrometry, demonstrating the fluorescence quenching effect of water, which is often used as a solvent. It was found that by increasing the amount of the organic phase in the DOX solvent the dynamic quenching is significantly suppressed. Ethanol, acetonitrile and dimethyl sulfoxide were tested and the best linearity of the calibration curve was obtained when above 50 % of the solvent was present in the binary mixture with water. Moreover, pH influence on the DOX fluorescence was also observed within the range of 4–10. Two times higher fluorescence intensity was observed at pH 4 compared to pH 10. Further, the DOX behavior in capillary electrophoresis (CE) was investigated. Electrophoretic mobilities (CE) in various pH of the background electrolyte were determined within the range from 16.3 to −13.3 × 10 −9 m−2 V−1 s−1. Finally, CE was also used to monitor the encapsulation of DOX into the cavity of apoferritin as well as the pH-triggered release.