Laser Induced Breakdown Spectroscopy (LIBS) was applied to homogenous methane–air mixtures of a wide range of compositions ranging from only air to only fuel in order to establish a new calibration scheme suitable for LIBS measurement of mixture fraction in turbulent non-premixed flames. Both a portable low resolution spectrometer equipped with a CCD detector with wide temporal detection window and a monochromator with a fast gated ICCD camera were employed to monitor the plasma emission. The results obtained using the two detection systems were fully consistent, suggesting that LIBS can be used successfully with the CCD detector that is more suitable for industrial applications. From the spectroscopic analysis, it was shown that the ratio of the intensities of Hα (656.3 nm) over O (777.3 nm) (Hα/Ο) and of C2 (Δυ = 0, d3Πg –#x03B1;3Πu) over CN (Δυ = 0, Β2Σ+ – Χ2Σ+) (C2/CN) depend monotonically on the mole fraction of methane in the ranges of 0.0–0.3 and 0.3–1.0 respectively, therefore providing a new scheme for measurement of mixture fraction in non-premixed systems spanning the complete range of equivalence ratios. The technique was also applied to a swirling recirculating premixed flame and it was found that the equivalence ratio is successfully measured in both reactants and products. Based on the above, LIBS experiments were then carried out in turbulent axisymmetric non-reacting and reacting jets, and the mean mixture fraction determined by the aforementioned calibration curves was in good agreement with empirical correlations, while the rms measurement showed the expected trends, demonstrating hence the usefulness of the technique.