Mitochondrial membrane potential (ΔΨm) is a critical component of the protonmotive force that drives ATP synthesis and supports essential mitochondrial functions, including metabolite transport, ion homeostasis, and protein import. In the parasitic protist Trypanosoma brucei, ΔΨm regulation is uniquely adapted across life cycle stages to meet changing metabolic demands. In the insect-stage procyclic form (PF), ΔΨm is generated by a canonical electron transport system (ETS), while in the bloodstream form (BF), where complexes III and IV are absent, ΔΨm is maintained by the reverse operation of ATP synthase, consuming ATP to pump protons. In T. brucei evansi, which lacks mitochondrial DNA, the ATP synthase is unable to translocate protons, and ΔΨm is sustained solely by electrogenic ADP/ATP exchange through the mitochondrial carrier. This chapter presents three complementary fluorescence-based methods to evaluate ΔΨm in T. brucei and T. b. evansi cells, highlighting their applicability to both intact and permeabilized parasites. We detail the use of two ΔΨm-sensitive dyes—TMRE, a cell-permeable dye suited for live-cell assays, and Safranine O, used in permeabilized preparations—and describe protocols for flow cytometry and fluorescence spectroscopy, respectively. These approaches allow robust, qualitative and semi-quantitative analysis of ΔΨm under different metabolic and experimental conditions. We address specific challenges associated with using fluorescent dyes to measure ΔΨm including issues of dye concentration, cellular permeability and potential artifacts that can affect interpretation of ΔΨm measurements.