© 2021, Hacettepe University, Faculty of Pharmacy. All rights reserved.Determination of the in vitro cytotoxic effects of an active drug substance, cosmetic product, environmental chemical (pesticides, endocrine disruptors, heavy metals, nanoparticles, etc.), and physical and biological agents has gained importance and popularity in the recent years. There are many methods in order to determine cytotoxicity or cell viability. These methods can be classified as follows: (i) dye exclusion methods; (ii) colorimetric methods; (iii) flourometric methods; (iv) luminometric methods; (v) determination of apoptosis by several techniques and (vi) determination of autophagy by different techniques. The researcher should evaluate all available methods using scientific data and select the most appropriate method. While making this evaluation, the physical and chemical properties of the test substance, the cell death mechanism by which the substance causes cytotoxicity and the specificity and sensitivity of the method should be considered. By this way, the reliability and accuracy of the result must be assured. In this review, the most frequently used methods for the determination of in vitro cytotoxicity have been explained and the advantages and disadvantages of these methods have been discussed.