Three-dimensional cultivation of human cells is promising especially for long-term maintenance of specific functions and mimicking the in vivo tissue environment. However, direct viability assessment is very difficult in such systems. Commonly applied indirect methods such as glucose consumption, albumin or urea production are greatly affected by culture conditions, stress and time of cultivation and do not reflect the real time viability of the cells. In this study we established a real-time in situ viability assay namely; resazurin assay, in a 3D hollow-fiber bioreactor using human liver cells. Resazurin assay is based on the conversion of resazurin to a fluorescent dye by cytoplasmatic and mitochondrial enzymes. We show that the resazurin reagent in concentrations used in this study is non-toxic and could be rapidly removed out of the system. Moreover, we observed that dead cells do not affect the results of the assay. We optimized the assay on HepG2 cells and tested it with primary human hepatocytes. Moreover, we maintained primary human hepatocytes in the 3D bioreactor system in serum-free conditions and also assessed viability before and after the exposure to amiodarone using the resazurin assay. We show that this approach is applicable during long-term cultivation of cells in bioreactors under different conditions and can moreover be applied to pharmacological studies, e.g. investigation of chronic drug effects in such 3D bioreactors.