The primary purpose of these researches was to optimize single-cell protein (SCP) production process using Saccharomyces cerevisiae NCAIM Y.00200 and Kluyveromyces marxianus DSM 4908 strain, and then to analyse the changes in yield of single-cell protein final product using vitamin supplementation. To determine these values, the total sugar content of the fermentation medium, and the protein content of the yeast was determined. During our work, a particular attention was paid to the change of sugar content and yeast protein quantity. Besides, yield (Yx/s) values, typical of the whole fermentation, were also measured. Protein yield, as the final product of fermentation, featured the efficiency of our work. The results of our optimized trial settings that were considered as control, using S. cerevisiae NCAIM Y.00200 and K. marxianus DSM 4908 strains, were compared with the results of vitamin-supplemented fermentation processes. On this basis, we can say that during our trials vitamin supplementation did not influence the final product yield of processes. The counted protein yields during fermentation were between 0.4–0.7 g g−1.