As per 3R, in vitro cytotoxicity tests such as the Neutral Red Update (NRU) assay are routinely used as alternative toxicity tests to eliminate the use of animals for acute oral toxicity tests. The NRU assay is one of the most commonly used cytotoxicity assays for chemicals, pharmaceuticals, cigarette smoke condensate, plant extracts and medical device extracts. As per the ICCVAM protocol, this test is generally performed in BALB/c 3T3 cells. The modal number of chromosomes in BALB/c 3T3 cells is 78 with a range of 62 to 109. The stem line number is hypo-tetraploid which makes this a karyotypically unstable cell line. We are proposing the use of the A549 cell line which is a hypotriploid human lung adenocarcinoma epithelial cell line. The modal number of chromosomes in A549 cells is 66 with a range of 64 to 67 and can be obtained from American Type Culture Collection (ATCC). Also, as per some of the peer-reviewed literature, this lung cell line may be useful to understand the role of alveolar Type II cells in the drug or chemical delivery at the pulmonary epithelium which makes this work relevant to tobacco research. Since the ICCVAM protocol is designed for the BALB/c 3T3 cells, we have performed a comparative study using A549 cells. We tested nine chemicals, from the NTP database with known different modes of action, using the 96-well plate method. The IC20, IC50, and IC80 values were calculated for each chemical, where possible. Our results indicate that the A549 cells can be used in lieu of the BALB/c 3T3 cells for the Neutral Red Uptake Assay.