The extensive diversity of volatile organic compounds, along with their minor structural variations, presents significant challenges in the development of chemosensory-based biosensors. Previously, we generated sensor cells expressing insect odorant receptors (ORs) in Sf21 cells, demonstrating their potential as cell-based odorant sensor elements. However, it remains unclear whether the selectivity of cells expressing ORs in vitro for diverse compounds aligns with the receptor’s in vivo performance, aside from the response to target compounds. To address this, we assessed the ligand responses of sensor cells expressing ORs from Drosophila melanogaster using a high-throughput calcium imaging system. Our results demonstrate that in vitro receptor responses exhibit ligand selectivity comparable to in vivo conditions across different chemical categories. Broadly tuned OR-expressing sensor cells (Or13a, Or47a, and Or98a) displayed differential affinities, whereas the narrowly tuned Or56a-expressing sensor cells selectively responded to geosmin. Moreover, cell responses varied with subtle differences in chemical structure, including carbon chain length and functional group positioning. These findings provide valuable insights into insect OR–ligand interactions in vitro, demonstrating that receptor selectivity in sensor cells closely mirrors in vivo conditions. In addition to this consistency, our results highlight the subtle ligand differentiation capabilities of sensor cells enabling fluorescence-based visualization of receptor–ligand interactions.