Sentinel lymph node biopsy (SLNB) has been recognized as "the gold standard" for axillary staging in early breast cancer patients with clinically negative lymph nodes, resulting in significant morbidity decrease and quality of life improvement. This study aims to validate the performance of a newly developed handheld portable gamma camera (PGC) produced by Imagensys (Italy), in detecting and locating sentinel lymph nodes (SLNs) during the preoperative and intraoperative phases in breast cancer patients compared to conventional lymphoscintigraphy. Adult female patients with histologically confirmed breast cancer, candidates for surgery and SLNB, were prospectively enrolled in this open-label, pre-marketing clinical trial. All patients underwent pre- operative assessment using both the PGC and conventional lymphoscintigraphy. The performance of the two devices was compared using the Poisson regression model for incidence rate ratios (IRRs). The intrinsic sensitivity of the devices was compared using the Wilcoxon Ranked Sign Test. The utility of PGC during intra-operative procedures was also evaluated. The manoeuvrability of the devices was evaluated using operator-satisfaction questioner. Sixty-eight patients (median age 50 years, BMI 21.4) were enrolled, including two patients with bilateral breast cancer, who underwent SLNB on both axillae. The PGC demonstrated superior preoperative lymph node detection rate (IRR 8.01, 95% CI 6.11-10.50; p < 0.0001) and intrinsic device sensitivity (mean counts per second 409 ± 286 vs. 255 ± 1173 for conventional device, p = 0.0003) compared to the conventional gamma camera. Intra-operative assessment with PGC was performed in 62 patients and no additional lymph nodes were visualised. However, the conventional gamma camera demonstrated superior manoeuvrability (p < 0.0001). The PGC handheld gamma camera showed promising results for preoperative SLN assessment in patients with breast cancer. The limited manoeuvrability may be related to the operator's experience leading to higher inter-operator variability. Appropriate training and frequent use of nuclear medicine and surgical equipment could overcome this limitation.