Organic solvents are widely used in microbial fermentations to reduce gas stripping effects and capture hydrophobic or toxic compounds. Reliable quantification of biochemical products in these overlays is highly challenging and practically difficult. Here, we present a significant improvement of identification and quantification methods for farnesene produced by Saccharomyces cerevisiae in two-liquid-phase fermentations using GC–MS and GC–FID. By increasing the polarity of the stationary phase introducing a ZB-50 column (50%-phenyl-50%-dimethylsiloxane) peak intensity could be increased and solvent car-ryover could be minimized. Direct quantification of farnesene in dodecane was achieved by GC–FID whereas GC–MS demonstrated to be an excellent technique for identification of known and unknown metabolites. The GC–FID is a suitable technique for direct quantification of farnesene in complex matrices as shown by the good calibration curve (R 2 40.998, N¼ 5) within the tested concentration range of 1– 50 mg/mL and the reproducibility of the intensity (intraday; o10% RSD at each concentration; N¼ 5). The limit of detection (LOD) and limit of quantification (LOQ) of the method were 0.24 and 0.80 mg/mL, respectively. Furthermore, the FID method proved to be highly stable with regard to the intensity of the calibration (N¼ 6) when the measurements were performed across 250 samples that were derived from a dodecane overlay.