Background and purpose: Despite being a critical treatment modality, radiotherapy effectiveness is often limited by tumor resistance. Therefore, there exists a need to identify molecular targeted drugs that enhance the therapeutic response to radiation. We hypothesize that a systematic comparison of targeted radiosensitizers across two-dimensional (2D) and three-dimensional (3D) spheroid cultures will reveal context-specific differences in radiosensitivity to guide preclinical prioritization of candidate radiosensitizers. Material and methods: Radiosensitizing effects of DNA-PKcs (M3814), ATR (M6620), PARP (Olaparib), and IAP (Birinapant) inhibitors using a panel of lung cancer cell lines were studied. A 3D extracellular matrix (ECM) colony formation assay for single doses of 0–6 Gy, coupled with automated colony counting, was implemented alongside standard 2D colony formation assays. Dose Enhancement Factor (DEF_0.1SF ) was used to compare radiosensitizing effects, and drug–radiation interactions were assessed using the Synergyfinder tool. Results: DNA-PKcs and ATR inhibitors induced moderate to strong dose-dependent radiosensitization (DEF_0.1SF > 1.4 for at least one drug concentration) in most cell lines under both conditions (15/16 drug/ cell line combinations). PARP inhibition showed similar effects in 3D and 2D (2/3 vs 3/5 combinations). Birinapant showed no relevant effect. The strongest synergy was at 2 Gy, particularly with the DNA-PK inhibitor in both culture models. Interpretation: Integrating multiple culture models enhances the detection of cell line – and drug-specific radiosensitization. Although 2D and 3D cultures produced largely similar results, and 2D assays provide a practical alternative when 3D methods are not feasible, the 3D cultures reveal additional ECM-dependent responses. These results emphasize the utility of physiologically relevant platforms for robust screening and prioritization of candidate radiosensitizers.