L-Carnitine is a naturally occurring substance required in mammalian energy metabolism that functions by facilitating long-chain fatty acid entry into cellular mitochondria, thereby delivering substrate for oxidation and subsequent energy production. It has been purposed that L-carnitine may improve and preserve cognitive performance, and may lead to better cognitive aging through the life span, and several controlled human clinical trials with L-carnitine support the hypothesis that this substance has the ability to improve cognitive function. We further hypothesized that, since L-carnitine is an important co-factor of mammalian mitochondrial energy metabolism, acute administration of L-carnitine to human tissue culture cells should result in detectable increases in mitochondrial function. Cultures of SH-SY-5Y human neuroblastoma and 1321N1 human astrocytoma cells grown in 96-well cell culture plates were acutely administered L-carnitine hydrochloride, and then, mitochondrial function was assayed using the colorimetric 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt cell assay kit in a VERSAmax tunable microplate reader. Significant increases in mitochondrial function were observed when human neuroblastoma or human astrocytoma cells were exposed to 100 nM (20 μg L-carnitine hydrochloride/L) to 100 μM (20 mg L-carnitine hydrochloride/L) concentrations of L-carnitine hydrochloride in comparison to unexposed cells, whereas no significant positive effects were observed at lower or higher concentrations of L-carnitine hydrochloride. The results of the present study provide insights for how L-carnitine therapy may significantly improve human neuronal function, but we recommend that future studies further explore different derivatives of L-carnitine compounds in different in vitro cell-based systems using different markers of mitochondrial function.