Adoptive cell therapy (ACT) with ex vivo expanded tumour-infiltrating lymphocytes (TILs) in combination with IL-2 is an effective treatment for metastatic melanoma. Modified protocols of cell expansion may allow the treatment of most enrolled patients and improve the efficacy of adoptively transferred cells. The aims of this study were to establish and validate the novel ‘Young TIL’ method at our institution and perform a head-to-head comparison of clinical-grade products generated with this protocol opposed to the conventional ‘Standard TIL’, which we are currently using in a pilot ACT trial for patients with melanoma. Our results confirm that ‘Young TILs’ display an earlier differentiation state, with higher CD27 and lower CD56 expression. In addition, CD8+ TILs expressing CD27 had longer telomeres compared with the CD27−. A recently described subset of NK cells, endowed with a high expression of CD56 (CD56bright), was detected for the first time in both types of cultures but at a higher frequency on Young TILs. Young and Standard TILs’ reactivity against autologous tumours was similar, with significant expression of TNF-α/IFN-γ/CD107a by CD8+ TILs detected in all cultures analysed. However, either slow expansion with high-dose IL-2 only or large numerical expansion with a rapid expansion protocol, which is required for current therapeutic protocols, significantly modified TIL phenotype by reducing the frequency of less differentiated, cancer-specific TILs. These studies further support the adoption of the Young TIL method in our current ACT trial and highlight the importance of continuous quality control of expansion protocols.