The determination of cell viability is essential to many areas of life sciences and biotechnology. Typically, cell viability measurements are based on the optical analysis of stained cells, which requires additional labeling steps and is hard to implement online. Frequency-dependent impedance flow cytometry (IFC) provides a label-free, fast, and reliable alternative to determine cell viability at the single cell level based on the Coulter principle. Here, we describe the application of IFC to eukaryotic cell cultures and compare the results to commonly used staining methods. Yeast cell parameters were assessed in normal and heat-inactivated cells as well as in alcoholic fermentation and long-term batch cultures providing a precise and fast determination of the cell viability and further quantitative measures of the cell culture status. As an important new application, we have investigated recombinant protein production in the widely used baculovirus insect cell expression system. The IFC analysis revealed the presence of a subpopulation of cells, which correlates with the protein expression yield, but it is not detectable with conventional optical cell counters. We tentatively identify this subpopulation as cells in the late phase of infection. Their detection can serve as a predictor for the optimal time point of harvest. The IFC technique should be generally applicable to many eukaryotic cell cultures in suspension, possibly also implemented online.