Introduction Holland PTC (Delft, The Netherlands) is an independent outpatient clinic where ten different types of cancer are treated with Pencil Beam Scanning (PBS). Some of these cancer types are situated in the thoracic region and therefore exposed to breathing motion. The interplay between the dynamic pencil beam delivered in PBS and the target motion in the thoracic region can lead to severe dose inhomogeneities. While interplay mitigation tactics, such as repainting and using larger spot sizes, are available, they can come at a dosimetric cost. Simulating the interplay effect aids in formulating clinical protocols for managing interplay-related challenges during treatment planning, as well as assessing the robustness of treatment plans both retrospectively and prospectively. Goals The primary goal is to perform clinical evaluation of the treatment beams of 18 patients’ treatment plans. Clinical evaluation can be achieved using an interplay simulation script. The secondary goal is to assess which patient-specific factors affect the interplay effect in these patients. Methods A derivative script, based on a clinically validated interplay simulation script, has been used to perform clinical evaluation. This derivative script allowed the input of 2-dimensional tumor motion rather than only superior-inferior (SI) tumor motion. 2- dimensional tumor motion information has been extracted from 18 lung cancer, esophagus cancer, and lymphoma patients. Quality Assurance (QA) dosimetric measurements were available of all the patients’ beams, 161 in total. The static QA dose measurements are compared to the same measurements with a two-dimensional sinusoidal motion in an in-house developed interplay simulation script. Simulations of each treatment beam consisted of a number of patient-specific fractions, with 100 treatment simulations performed for each beam. Gamma acceptance criteria are set at 2% dose difference and 2mm distance to agreement. The last fraction where a treatment plan is below the predefined robustness threshold, known as the Last Fraction below Threshold (LFBT), is the metric on which clinical evaluation is based. If the 90th percentile LFBT was equal to or below the number of prescribed treatment fractions, a treatment beam is considered robust against the interplay effect. Near the end of thesis completion, the discovery was made that the interplay simulation script used throughout the majority of this thesis yielded results that differed notably and clinically from those generated by the validated script. Due to the work and time needed to repeat the simulations with the correct script, and the time necessary to analyze and interpret the new results, the decision was made not to repeat the simulations for all beams with the validated script. The simulations are repeated for nine out of 161 beams to compare the outcomes of both scripts. Results Using the unvalidated script,tumor-type specific interplay analysis shows interplay robustness in tumors with motion amplitude of up to 9 mm in lymphomas, 5 mm in lung cancer patients, and 6 mm in esophageal cancer patients. A high correlation (up to r=0.95) between 2D tumor motion amplitude and interplay robustness was found. The clinically validated script’s results show that all nine treatment beams are robust against the interplay effect, even when the amplitude is 11.22 mm in SI direction. All the patient beams with a tumor motion amplitude greater than 8.45 mm in SI direction are not robust according to the unvalidated script. This suggests that the unvalidated script underestimated the interplay robustness of the treatment beams. Implications/Conclusion Since only 9 out of 161 beams have been simulated using this script, limited general conclusions can be drawn from this data. The results of the clinically validated script suggest all beams up to 11.22 mm tumor motion amplitude are robust against the interplay.