Much of our understanding of protein folding mechanisms is derived from experiments using intrinsic fluorescence of natural or genetically inserted tryptophan (Trp) residues to monitor protein refolding and site-directed mutagenesis to determine the energetic role of amino acids in the native (N), intermediate (I) or transition (T) states. However, this strategy has limited use to study complex folding reactions because a single fluorescence probe may not detect all low-energy folding intermediates. To overcome this limitation, we suggest that protein refolding should be monitored with different solvent-exposed Trp probes. Here, we demonstrate the utility of this approach by investigating the controversial folding mechanism of ubiquitin (Ub) using Trp probes located at residue positions 1, 28, 45, 57, and 66. We first show that these Trp are structurally sensitive and minimally perturbing fluorescent probes for monitoring folding/unfolding of the protein. Using a conventional stopped-flow instrument, we show that ANS and Trp fluorescence detect two distinct transitions during the refolding of all five Trp mutants at low concentrations of denaturant: T(1), a denaturant-dependent transition and T(2), a slower transition, largely denaturant-independent. Surprisingly, some Trp mutants (Ub(M1W), Ub(S57W)) display Trp fluorescence changes during T(1) that are distinct from the expected U-->N transition suggesting that the denaturant-dependent refolding transition of Ub is not a U-->N transition but represents the formation of a structurally distinct I-state (U-->I). Alternatively, this U-->I transition could be also clearly distinguished by using a combination of two Trp mutations Ub(F45W-T66W) for which the two Trp probes that display fluorescence changes of opposite sign during T(1) and T(2) (Ub(F45W-T66W)). Global fitting of the folding/unfolding kinetic parameters and additional folding-unfolding double-jump experiments performed on Ub(M1W), a mutant with enhanced fluorescence in the I-state, demonstrate that the I-state is stable, compact, misfolded, and on-pathway. These results illustrate how transient low-energy I-states can be characterized efficiently in complex refolding reactions using multiple Trp probes.