Simple, rapid, and efficient cell viability assays play a fundamental role in much of biomedical research, including cell toxicology investigations and antitumor drug screening. Here, we demonstrate for the first time a rapid and label-free cell viability assay using THz spectroscopy in combination with a new optical clearing agent (OCA) and microfluidic technology. This strategy uses a considerably less absorptive OCA to replace the highly absorptive water molecules around the living cells and thus to decrease the background signal interference. Three low-viscosity oils were screened as potential OCA candidates, among which fluorinated oil was selected because of its lower absorption and lowest cytotoxicity. After the liquid medium was replaced with fluorinated oil in a microfluidic chip, an obvious THz spectral difference was observed between the fluorinated oils with and without living cells. This change in THz response was preliminarily attributed to the distinguishable signals between the cells and the fluorinated oil. In addition, we applied this method to cell viability assays of human breast cancer cells (MDA-MB-231) after treatment with different antitumor drugs. The results indicated that THz spectroscopy with the aid of the proposed water-replacement strategy presented excellent quantification of cell viability with the advantages of a rapid, label-free, nondestructive microassay, which offers significant potential to developing a convenient and practical cell analysis platform.