Abstract: A method of testing a test substance in an in vitro model of a human tracheobronchial respiratory tract, includes: providing the in vitro model including a cell culture including airway epithelial cells (AECs), a basolateral compartment below the AECs and an apical compartment above the AECs, wherein the AECs form a barrier between the basolateral AND apical compartments; adding the test substance to the apical and/or basolateral compartment; adding a tracer to the basolateral compartment, which is fluorescent, has a molecular weight within 5 kDa of human albumin and is added before, during or after adding the test substance; incubating the cell culture system in a presence of the tracer; collecting at least one sample from the apical compartment; and measuring a fluorescence thereof to determine an effect of the test substance on the AECs. A kit is also disclosed.