Since 1991, the NAD(P)H-aided conversion of resazurin to fluorescent resorufin has been widely used to measure viability based on the metabolic activity in mammalian cell culture and primary cells. However, different research groups have used divergent assay protocols, scarcely reporting the systematic optimization of the assay. Here, we perform extensive studies to fine-tune the experimental protocols utilizing resazurin-based viability sensing. Specifically, we focus on (A) optimization of the assay dynamic range in individual cell lines for the correct measurement of cytostatic and cytotoxic properties of the compounds; (B) dependence of the dynamic range on the physical quantity detected (fluorescence intensity versus change of absorbance spectrum); (C) calibration of the assay for the correct interpretation of data measured in hypoxic conditions; and (D) possibilities for combining the resazurin assay with other methods including measurement of necrosis and apoptosis. We also demonstrate the enhanced precision and flexibility of the resazurin-based assay regarding the readout format and kinetic measurement mode as compared to the widely used analogous assay which utilizes tetrazolium dye MTT. The discussed assay optimization guidelines provide useful instructions for the beginners in the field and for the experienced scientists exploring new ways for measurement of cellular viability using resazurin.