Colchicine (COL) is a bioactive molecule with antitumor properties. When COL binds to tubulin (TU), it inhibits microtubule assembly dynamics. We have investigated COL–TU interactions using laser flash photolysis (LFP) technique and performing fully flexible molecular dynamics simulations. Excitation of COL at 355 nm in aqueous medium did not lead to any transient absorption spectrum. By contrast, in the presence of TU a transient peaking at λ_max ca. 420 nm was registered and assigned as triplet excited COL complexed with TU (³COL*@TU). In aerated medium, the lifetime was τ ca. 160 μs and the quantum yield was 0.138. Likewise, when the bicyclic COL analog MTC was submitted to LFP in the presence of TU, ³MTC@TU* was detected with a lifetime of ca. 62 μs and a quantum yield of 0.296, Aqueous solutions of MTC did not produce any signal in the microsecond timescale. The triplet energy of MTC was obtained by means of emission measurements and found to be ca. 200 kJ mol⁻¹, a value that matches with that previously reported for COL (188 kJ mol⁻¹). Molecular dynamic simulations, both with the ground and triplet excited state, reveal a strong interaction between COL and TU to give stabilized complexes with restricted mobility inside the protein binding site. These results demonstrate that LFP is a useful methodology to study the binding of COL derivatives to TU and open a new way to evaluate the interactions of non-fluorescent anticancer drugs with this protein.