Bistable [2]rotaxanes in which the affinities of the two stations can be reversed form the basis of molecular shuttles. Gaining quantitative information on such rotaxanes in which the ring distribution between the two stations is largely nonsymmetric has proven to be very challenging. Herein, we report on two independent experimental methodologies, based on luminescence lifetime measurements and acid–base titrations, to determine the relative populations of the two co‐conformations of a [2]rotaxane. The assays yield convergent results and are sensitive enough to measure an equilibrium constant (K≈4000) out of reach for NMR spectroscopy. We also estimate the ring distribution constant in the switched (deprotonated) state (K′<10⁻⁴), and report the highest positional efficiency for stimuli‐induced shuttling to date (>99.92 %). Finally, our results show that the pK_a of the pH‐responsive station depends on the ring affinity of the pH‐insensitive station, an observation that paves the way for the design of new artificial allosteric systems.