<p>Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease of motor neurons, leading to fatal muscle paralysis. Familial forms of ALS (fALS) account for approximately 10% of cases. Alterations of mitochondrial functions have been proposed to contribute to disease pathogenesis. Here, we employed a direct conversion (DC) technique to generate induced motor neurons (iMN) from skin fibroblasts to investigate mitochondrial phenotypes in a patient-derived disease relevant cell culture system. We converted 7 control fibroblast lines and 17 lines harboring the following fALS mutations, SOD1^A4V, TDP-43^N352S, FUS^R521G, CHCHD10^R15L, and C9orf72 repeat expansion. We developed new machine learning approaches to identify iMN, analyze their mitochondrial function, and follow their fate longitudinally. Mitochondrial and energetic abnormalities were observed, but not all fALS iMN lines exhibited the same alterations. SOD1^A4V, C9orf72, and TDP-43^N352S iMN had increased mitochondrial membrane potential, while in CHCHD10^R15L cells membrane potential was decreased. TDP-43^N352S iMN displayed changes in mitochondrial morphology and increased motility. SOD1^A4V, TDP-43^N352S, and CHCHD10^R15L iMN had increased oxygen consumption rates and altered extracellular acidification rates. FUS^R521G mutants had decreased ATP/ADP ratio, suggesting impaired energy metabolism. SOD1^A4V, C9orf72, and TDP-43^N352S had increased, while FUS^R521G had decreased mitochondrial reactive oxygen species production. We tested the viability of iMN and found decreases in survival in SOD1^A4V, C9orf72, and FUS^R521G, which were corrected by small molecules that target mitochondrial stress and worsened by bioenergetic stressors. Together, our findings reinforce the role of mitochondrial dysfunction in ALS and indicate that fibroblast-derived iMN may be useful to study fALS metabolic alterations. Strengths of the DC iMN approach include low cost, speed of transformation, and the preservation of epigenetic modifications. However, further refinement of the fibroblasts DC iMN technique is still needed to improve transformation efficiency, reproducibility, the relatively short lifespan of iMN, and the senescence of the parental fibroblasts.</p>