In the advanced stages of non-small cell lung cancer (NSCLC), molecular testing is often performed on archival cytological smears. The nCounter system (NanoString Technologies) is a new promising multiplex digital colour-coded barcode technology. However, its feasibility to evaluate the RNA expression of clinical relevant biomarkers on routine cytological smears is still uncertain. To this end, RNA was extracted from 12 NSCLC routine stained cytological smears, and nCounter analysis performed by using a 48-gene panel. Overall, 11/12 (92%) of the smears were adequate for the secondary analysis, fulfilling the quality check parameter analysis of nSolver software. This pilot study shows that RNA nCounter analysis is feasible on routine cytological smears preparing the field for the implementation of this technology in the routine setting.