Background/Objectives: Surgical access and one-lung ventilation (OLV) may generate distinct systemic and local inflammatory responses after lung resection. We compared perioperative cytokine profiles in serum, pleural fluid, and bronchoalveolar lavage (BAL) after video-assisted thoracoscopic surgery (VATS) and open thoracotomy for non-small cell lung cancer (NSCLC). Methods: This prospective, single-center, non-randomized observational study included 40 adults undergoing anatomical lobectomy with mediastinal lymph-node dissection (VATS, n = 20; open thoracotomy, n = 20). Surgical access was selected according to patient preference. TNF-α, IL-6, IL-8, and IL-10 were measured in serial serum and pleural-fluid samples and in BAL from both lungs before OLV and after resection. Results: Serum and pleural-fluid cytokines peaked at 4 h in both groups. Exploratory pointwise comparisons showed that at 24 h, serum TNF-α (4.89 [IQR, 4.43–5.92] vs. 4.01 [IQR, 3.75–5.12] pg/mL; p = 0.010), IL-6 (150.22 [IQR, 136.90–194.66] vs. 108.00 [IQR, 100.46–125.88] pg/mL; p < 0.001), and IL-8 (66.83 [IQR, 57.70–82.82] vs. 43.62 [IQR, 32.89–50.59] pg/mL; p = 0.001) were higher after thoracotomy than after VATS; differences at 48 h persisted for TNF-α (2.48 [IQR, 1.86–3.04] vs. 1.99 [IQR, 1.19–2.23] pg/mL; p = 0.010) and IL-8 (16.92 [IQR, 15.09–23.99] vs. 7.42 [IQR, 6.44–11.30] pg/mL; p < 0.001). Pleural-fluid IL-6 at 24 h and IL-8 at 48 h were also higher after thoracotomy, whereas BAL cytokines did not differ significantly by approach. Longitudinal GEE analyses showed significant approach-by-time interactions for serum TNF-α, IL-6, and IL-8 and for pleural-fluid IL-8, but not for pleural-fluid IL-6. Conclusions: VATS was associated with a lower systemic proinflammatory response than open thoracotomy, while differences in the pleural compartment were more limited and no detectable difference was observed in early alveolar cytokine responses. These findings highlight compartment-specific differences in perioperative inflammatory responses between VATS and open thoracotomy.