The proper functioning of mitochondria requires preserving the membrane potential (ΔΨm) within a narrow window. Significant deviation from the membrane potential of mitochondria is a well-established indicator of mitochondrial dysfunction. Various pathological conditions, such as cancer, diabetes, and cardiotoxicity, have been linked to mitochondrial dysfunction, highlighting the need for reliable methods to assess membrane potential in vivo. Hence, there is a need to explore radiolabeled lipophilic cations that accumulate within mitochondria in proportion to the potential gradient. The lipophilic organic cation triphenylphosphonium (TPP) has gained attention as a promising tracer for non-invasive imaging of mitochondrial function. It has been labeled with various radioisotopes, such as F-18, for imaging mitochondrial membrane potential in cancer through positron emission tomography (PET). The first attempt to quantify membrane potential in living organisms by examining the biodistribution of the ¹¹C-labeled TPP derivative was reported more than 30 years ago. Herein, a series of TPP derivatives, together with TPP, have been radiolabeled with ⁶⁴Cu for imaging gliomas, which are characterized by high mitochondrial content. 2-(diphenylphosphoryl)-ethyldiphenylphosphonium (TPEP) has demonstrated superior tumor uptake and favorable tumor-to-background ratios, leading to its selection for further assessment as a magnetic resonance imaging contrast agent. Building on these findings, we developed ⁶⁸Ga-labeled TPP and TPEP as novel PET tracers for rhabdomyosarcoma. To assess how the choice of targeting moiety and bifunctional chelator influences tracer performance, biodistribution studies were conducted in mitochondrial-rich rhabdomyosarcoma patient-derived xenografts. These results support further development of ⁶⁸Ga-TPP-based agents for mitochondrial-targeted oncologic imaging applications.