Background: 3′-Deoxy-3′-[18F]fluorothymidine ([18F]FLT) was proposed as an imaging biomarker for the assessment of in vivo cellular proliferation with positron emission tomography (PET). The current study aimed to validate [18F]FLT as a perfusion-independent PET tracer, by gaining insight in the intra-tumoural relationship between [18F]FLT uptake and perfusion in non-small cell lung cancer (NSCLC) patients undergoing treatment with a tyrosine kinase inhibitor (TKI). Six patients with metastatic NSCLC, having an activating epidermal growth factor receptor (EGFR) mutation, were included in this study. Patients underwent [15O]H2O and [18F]FLT PET/CT scans at three time points: before treatment and 7 and 28 days after treatment with a TKI (erlotinib or gefitinib). Parametric analyses were performed to generate quantitative 3D images of both perfusion measured with [15O]H2O and proliferation measured with [18F]FLT volume of distribution (VT). A multiparametric classification was performed by classifying voxels as low and high perfusion and/or low and high [18F]FLT VT using a single global threshold for all scans and subjects. By combining these initial classifications, voxels were allocated to four categories (low perfusion-low VT, low perfusion-high VT, high perfusion-low VT and high perfusion-high VT). Results: A total of 17 perfusion and 18 [18F]FLT PET/CT scans were evaluated. The average tumour values across all lesions were 0.53 ± 0.26 mL cm− 3 min− 1 and 4.25 ± 1.71 mL cm− 3 for perfusion and [18F]FLT VT, respectively. Multiparametric analysis suggested a shift in voxel distribution, particularly regarding the VT: from an average of ≥ 77% voxels classified in the “high VT category” to ≥ 85% voxels classified in the “low VT category”. The shift was most prominent 7 days after treatment and remained relatively similar afterwards. Changes in perfusion and its spatial distribution were minimal. Conclusion: The present study suggests that [18F]FLT might be a perfusion-independent PET tracer for measuring tumour response as parametric changes in [18F]FLT uptake occurred independent from changes in perfusion. Trial registration: Nederlands Trial Register (NTR), NTR3557. Registered 2 August 2012.