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In some fermentation systems, whey components (lactose, proteins and minerals) can produce isopentyl acetate (IA). An analysis of the best conditions for IA production with Kluyveromyces marxianus was developed in this work. The experiment design was two-factor and three-level design based on a response surface methodology (RSM) using Design-Expert® software. The analysis of anomeric protons by nuclear magnetic resonance (1H-NMR) showed 81.25% of β lactose content. This characteristic favored the production of IA. The maximum output (Mp) of IA, determined by gas chromatography, was 9.52 g/L (p < 0.05). The central composite design (CCD) was used to perform the factor analysis. Results showed that concentrations of 0.03 (g/L) ammonium sulphate and 0.3 (v/v) of isoamyl alcohol are the best conditions for a maximum rate of IA production. The production of IA can reduce the discharge of whey, allowing its reuse and revaluation.