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Since the advent of the acellular pertussis vaccine, safety testing has been carried out using the Histamine Sensitisation Assay (HIST assay). This assay is crude in nature and involves large numbers of mice to ensure statistically relevant output. In this work, a permeability assay is described that is a viable alternative to the HIST assay. Human Umbilical Vein Endothelial Cells (HUVECs) in co-culture with peripheral blood mononuclear cells (PBMCs) were used in a permeability assay to distinguish a preparation of DTaP5-IPV-Hib vaccine spiked with pertussis toxin (PTx) from a second control preparation. Using this assay, permeability of the HUVEC monolayer was determined to be a reliable indicator of PTx activity. TNF-α secretion was quantified to determine if there was a correlation with permeability, however, it was highly variable between blood donors. Therefore, it was likely that the permeability was the result of direct interaction between PTx and the HUVECs. Tight junctional (TJ) dysregulation as demonstrated by immunostaining the TJ complex and measuring the transendothelial electrical resistance (TEER) was investigated as the primary cause of PTx-induced permeability. Subsequent investigation into gap junction functionality corroborated this as a reduction in connexin functionality was observed which can be partially explained by TJ dysfunction, however, PTx was also shown to impair connexin 43 deposition at the plasma membrane, demonstrating that TJ expression could be used as alternative and/or combination assay. Additionally, Fluorescence Lifetime Imaging (FLIM) was carried out to directly image PTx activity within the HUVECs, by imaging endogenous NADH fluorescence. PTx activity was shown to be associated with a small increase in protein-bound NADH and since FLIM is non-destructive this allowed other experiments to be carried out on the same generation of HUVECs. In conclusion, the permeability assay described here is capable of detecting PTx in vaccine preparations at concentrations below the required threshold of 4 IU/ml PTx.