**1. Introduction**

Cytotoxicity assays measure the destructive capacity of substances on living cells or tissue systems. These assays play a key role in the biomedical field as they provide information on the therapeutic potential of the biochemical molecules. Cytotoxicity assays can be applied to both *in vivo* and *in vitro* systems where each method has some advantages and disadvantages. In this chapter, we present and discuss conventional *in vitro* colorimetric cytotoxicity assays that were optimized for spectrophotometric analysis.

There are many cytotoxicity tests with different mechanisms and sensitivities. However, cytotoxicity determination methods are generally examined in three groups. These are colorimetric, luminescence, and enzymatic methods. Luminometric methods are divided into fluorescence and bioluminescence. Fluorometric assays utilize fluorescent substances such as resazurin and are performed with a fluorometer or fluorescence microplate reader. On the other hand, cytotoxicity detection in bioluminescent methods is made by an enzyme called luciferase. In addition, in the real-time bioluminescence method, the exposure of cells to cytotoxic substances has become possible to be examined during the exposure. Enzymes leaking into the medium following cell damage or death have also been considered markers of dead cell counts. Among these enzymes, lactate dehydrogenase (LDH), which stands out with its stability, has taken its place among viability tests as a marker of cell death.

Colorimetric methods are techniques based on color change using tetrazolium salts or specific staining of cells using crystal violet and neutral red dyes. The first colorimetric method was described by Mossman et al., in 1983, which was developed as an alternative to the labor-intensive, time-consuming, and costly radioactive methods of measuring surviving and/or proliferating mammalian cells [1]. Furthermore, they can measure large numbers of samples with a high degree of precision directly in the plate by using a spectrophotometer or plate reader [2, 3]. There are also methods used especially in routine cell culture processes, such as microscopy (staining with trypan blue-Thoma slide) or an automatic cell counter that can be used in cytotoxicity studies. However, these methods are very time-consuming and are not suitable for studies with a large number of samples.

The basic principle of colorimetric assays lies in the color development as a result of metabolic activity rate of the cells. When a reagent meets with the viable cells, it is converted to a colored product by the metabolic activity of the cells. Such color change can easily be detected, measured, and quantified via spectrophotometric measurements as absorbance at a specific wavelength (**Figure 1**). Collected signal reflects the viable cell population or metabolic activity of the cells. As dying cells lose their function to convert substrate into a detectable colored product, the observed signal would be less than the active population.

#### **Figure 1.** *A simple representation of colorimetric detection assays.*
