Background: Metastatic UM is a rare cancer with poor response rates to systemic therapy. Adoptive transfer of patient-specific TIL may represent the best strategy for treatment. TIL are harvested from primary or metastatic tumors and initially expanded in culture with high dose IL-2 prior to undergoing rapid expansion protocol and therapeutic administration. Here, we report improved rates of initial expansion using a previously described TIL 3.0 method which utilizes dual agonistic antibodies to TCR and 4-1BB (Urelumab) for stimulation, respectively, with high dose IL-2, compared to the traditional method. Methods: Between 2006 and 2019, patients were consented for TIL harvest from either primary or metastatic UM tumors. Demographics, clinical features, and outcomes of the TIL initial expansion were collected. Success rates, number of cells expanded, and days in culture for the two methods were analyzed using partially overlapping samples t-tests and z-tests. Results: There were 85 harvests and expansions from 76 patients using the traditional method and 32 expansions from 30 patients using TIL 3.0. Initial TIL expansion was successful in 97% of TIL 3.0 harvests compared to 35% for the traditional method (p < 0.001). More TIL were expanded with TIL 3.0 compared to the traditional method (291.3 million cells vs. 88.6 million cells, p < 0.001), and fewer days were required in culture (18.5 vs. 29.0, p < 0.001). Both primary UM harvests and metastatic harvests were more successful with TIL 3.0 (90% vs. 12% for primary, p < 0.001, and 100% vs. 42% for metastatic, p < 0.001). Conclusions: Expansion of UM tumors via the TIL 3.0 method led to successful growth in 97% of harvests. Therapeutic administration to patients with TIL 3.0 is under active investigation.