A sensitive quantitative procedure for cytotoxicity assays using monolayer cultures is described. The method involves two steps: (1) microscopic screening for morphological changes after an experimental protocol and (2) quantification of surviving cells using neutral red, a supravital dye, incorporated into lysosomes of viable cells, followed by colorimetric analysis. Both assays are performed on the same cell culture in 96-well microtiter plates and can be automated with a spectrophotometric microplate reader. The test is adaptable to conventional spectrophotometers. The procedure avoids the need for trypsinization and trypan blue exclusion. It is suitable for screening cytotoxic agents and has been standardized for use with automated readers. The method uses 3T3 mouse fibroblasts, and cells are seeded at a density leading to 60-70% confluence after 24 hours. Test agents are diluted in medium and incubated for 24 hours. The highest tolerated dose (HTD) is determined by scoring morphological effects. The neutral red assay involves washing, adding dye, incubation, fixation, extraction, and spectrophotometric analysis. NR-90 is the endpoint, defined as 90% of control absorbance. NR-5 represents a highly toxic endpoint. The method is rapid, reliable, and reproducible, suitable for in vitro screening of cytotoxic agents. Key components include reagents, materials, and equipment for the procedure. The method provides a sensitive and quantitative approach for assessing cell viability and cytotoxicity in monolayer cultures.A sensitive quantitative procedure for cytotoxicity assays using monolayer cultures is described. The method involves two steps: (1) microscopic screening for morphological changes after an experimental protocol and (2) quantification of surviving cells using neutral red, a supravital dye, incorporated into lysosomes of viable cells, followed by colorimetric analysis. Both assays are performed on the same cell culture in 96-well microtiter plates and can be automated with a spectrophotometric microplate reader. The test is adaptable to conventional spectrophotometers. The procedure avoids the need for trypsinization and trypan blue exclusion. It is suitable for screening cytotoxic agents and has been standardized for use with automated readers. The method uses 3T3 mouse fibroblasts, and cells are seeded at a density leading to 60-70% confluence after 24 hours. Test agents are diluted in medium and incubated for 24 hours. The highest tolerated dose (HTD) is determined by scoring morphological effects. The neutral red assay involves washing, adding dye, incubation, fixation, extraction, and spectrophotometric analysis. NR-90 is the endpoint, defined as 90% of control absorbance. NR-5 represents a highly toxic endpoint. The method is rapid, reliable, and reproducible, suitable for in vitro screening of cytotoxic agents. Key components include reagents, materials, and equipment for the procedure. The method provides a sensitive and quantitative approach for assessing cell viability and cytotoxicity in monolayer cultures.