We aimed to determine a method for prescribing a standing prosthetic leg length (ProsL) that results in an equivalent running biological leg length (BioL) for athletes with unilateral (UTTA) and bilateral transtibial amputations (BTTA). We measured standing leg length of ten non-amputee (NA) athletes, ten athletes with UTTA, and five athletes with BTTA. All athletes performed treadmill running trials from 3 m/s to their maximum speed. We calculated standing and running BioL and ProsL lengths and assessed the running-to-standing leg length ratio (Lratio) at three instances during ground contact: touchdown, mid-stance, and take-off. Athletes with UTTA had 2.4 cm longer standing ProsL than BioL length (p = 0.030), but their ProsL length were up to 3.3 cm shorter at touchdown and 4.1 cm shorter at mid-stance than BioL, at 3–11.5 m/s. At touchdown, mid-stance, and take-off, athletes with BTTA had 0.01–0.05 lower Lratioat 3 m/s (p < 0.001) and 0.03–0.07 lower Lratioat 10 m/s (p < 0.001) in their ProsL compared to the BioL of NA athletes. During running, ProsL were consistently shorter than BioL. To achieve equivalent running leg lengths at touchdown and take-off, athletes with UTTA should set their running-specific prosthesis height so that their standing ProsL length is 2.8–4.5% longer than their BioL length, and athletes with BTTA should set their running-specific prosthesis height so that their standing ProsL lengths are at least 2.1–3.9% longer than their presumed BioL length. Setting ProsL length to match presumed biological dimensions during standing results in shorter legs during running.