In the differentiating culture of myoblasts, the formation of myofibrils is accompanied by the consumption of amino acids and the release of their derivatives into the medium. The enhancement of mitochondrial metabolism precedes the formation of myofibrils. In this work, the release of a humanin-like peptide (HNLP, mt-RNR-peptide) and amino acid derivatives into the medium was studied in the differentiation of C2C12 myoblasts. Cells of the C2C12 line were cultured using standard techniques in plates with control of myofibril formation and samples selection for analysis at 0 (induction), 2, 4, 7, 9 and 11 days of differentiation. HNLP was determined by enzyme immunoassay, and amino acids and their metabolites by liquid chromatography. From the essential amino acids, branched chain - Val, Leu, Ile were most intensively consumed up to day 7 of differentiation. There was observed a mutual correlation between the daily production of arginine derivatives - homoarginine (hArg) and ornithine (r = 0.53, p = 0.008) with a maximum on day 2, a significant decrease of hArg production on day 4 and trace secretion after day 7. The release of HNLP, unlike hArg, lasts up to 4 days. Secretion of HNLP in course differentiating myoblasts is an early marker of the mitochondrial metabolism development. Its decrease by day 7 is associated to inhibition of the further existence of already differentiated myocytes. The early stage of cell culture differentiation is more clearly checked not by the level of essential amino acids intake, which presented in the cultivation medium at high concentrations, but by the formation of amino acid products of specialized metabolic pathways, including hArg and ornithine. The metabolic activity of mitochondria is confirmed by the secretion of HNLP. The specific functional activity of myocytes depends on the metabolic activity of the mitochondria, which can be checked without violating the integrity of the culture, according to the level of secreted HNLP.