We apply our new approach of quantum Separation of Variables (SoV) to the complete characterization of the transfer matrix spectrum of quantum integrable lattice models associated to gl(n)-invariant R-matrices in the fundamental representations. We consider lattices with N sites and quasi-periodic boundary conditions associated to an arbitrary twist K having simple spectrum (but not necessarily diagonalizable). In our approach the SoV basis is constructed in an universal manner starting from the direct use of the conserved charges of the models, i.e., from the commuting family of transfer matrices. Using the integrable structure of the models, incarnated in the hierarchy of transfer matrices fusion relations, we prove that our SoV basis indeed separates the spectrum of the corresponding transfer matrices. Moreover, the combined use of the fusion rules, of the known analytic properties of the transfer matrices and of the SoV basis allows us to obtain the complete characterization of the transfer matrix spectrum and to prove its simplicity. Any transfer matrix eigenvalue is completely characterized as a solution of a so-called quantum spectral curve equation that we obtain as a difference functional equation of order n. Namely, any eigenvalue satisfies this equation and any solution of this equation having prescribed properties leads to an eigenvalue. We construct the associated eigenvector, unique up to normalization, by computing its decomposition on the SoV basis that is of a factorized form written in terms of the powers of the corresponding eigenvalues. If the twist matrix K is diagonalizable with simple spectrum then the transfer matrix is also diagonalizable with simple spectrum. In that case, we give a construction of the Baxter Q-operator satisfying a T-Q equation of order n, the quantum spectral curve equation, involving the hierarchy of the fused transfer matrices.