Photodynamic therapy (PDT) combines light, a photosensitizing chemical substance, and molecular oxygen to elicit cell death and is employed in the treatment of a variety of diseases, in cluding cancer. The development of PDT treatment strategies requires in vitro assays to develop new photosensitizers. One such assay is the MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide developed in 1983 and widely used in PDT studies. Despite the exponential growth in the number of publications, a uniform MTT protocol for use in the PDT area is lacking. Herein, we list and standardize the conditions to evaluate the photosensitizer methylene blue (MB) in glioblastoma and neuroblastoma cell lines. In addition, we review technical pitfalls and identify several variables that must be taken into consideration in order to provide accurate results with MTT. We conclude that for each cell line we must have a dose-response curve using the MTT assay and good controls for the standardization. Additionally, the optimal values of the time and cell density must be in the linear range of the curve to avoid errors. We describe all relevant points and outline the best normalization techniques to observe the differences between treatments.