The impact of nanomaterials such as carbon nanotubes on biological matter is a topic of increasing interest and concern and requires a multifaceted approach to be resolved. A modified cytotoxic (lactate dehydrogenase (LDH)) assay is developed in an attempt to offer a valid and reliable methodology for screening carbon nanotube toxicity in vitro. Two of the most widely used types of surface-modified multiwalled carbon nanotubes (MWNTs) are tested: ammonium-functionalized MWNTs (MWNT-NH 3 +) and Pluronic F127 coated MWNTs (MWNT:F127). Chemically functionalized MWNTs show significantly greater cellular uptake into lung epithelial A549 cells compared to the non-covalently Pluronic F127-coated MWNTs. In spite of this, MWNT:F127 exhibit enhanced cytotoxicity according to the modified LDH assay. The validity of the modified LDH assay is further validated by direct comparison with other less reliable or accurate cytotoxicity assays. These findings indicate the reliability of the modified LDH assay as a screening tool to assess carbon nanotube cytotoxicity and illustrate that high levels of carbon nanotube cellular internalization do not necessarily lead to adverse responses. A modified cytotoxic (LDH) assay is developed in an attempt to offer a valid and reliable methodology for screening carbon nanotube (CNT) toxicity in vitro. Two types of CNT are screened using this assay and the results are compared to other commonly used assays. This assay is a promising tool, particularly in a field eager for reliable in vitro cytotoxicity assays. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.