Rationale: Resection of soft tissue sarcomas (STS) is accompanied by a high rate of tumor-positive surgical margins (14-34%), which potentially leads to decreased disease-free survival. Vascular Endothelial Growth Factor-A (VEGF-A) is overexpressed in malignant tumors, including STS, and can be targeted with bevacizumab-800CW during fluorescence-guided surgery for real-time tumor detection. In this phase 1 clinical trial, we determined the feasibility, safety and optimal dose of bevacizumab-800CW for fluorescence-guided surgery (FGS) in STS for in- and ex vivo tumor detection. Methods: Patients with a histopathologically diagnosis of STS were included. In the dose-escalation phase, patients received bevacizumab-800CW intravenously 3 days prior to surgery (10, 25 and 50 mg, n = 8). In the subsequent dose-expansion phase, 7 additional patients received bevacizumab-800CW at the optimal dose. Fluorescence images in- and ex vivo were obtained during all stages of standard of care. The optimal dose was determined by calculating in- and ex vivo Tumor-to-Background ratios (TBR) and correlating these results with histopathology. Results: Fifteen patients with STS completed this study. All tumors could be visualized during in- and ex vivo imaging. The optimal bevacizumab-800CW dose proved to be 10 mg with a median in vivo TBR of 2.0 (±0.58) and an ex vivo TBR of 2.67 (±1.6). All seven tumor-positive margins could be observed real-time after surgical resection. Conclusion: FGS using 10 mg bevacizumab-800CW is feasible and safe for intra-operative imaging of STS, potentially allowing tumor detection and margin assessment during surgery. An additional follow-up phase II study is needed to confirm the diagnostic accuracy.