A relatively high level of stratification (qualitatively: lack of homogeneity) is one of the main advantages of partially premixed combustion over the homogeneous charge compression ignition concept. Stratification can smooth the heat release rate and improve the controllability of combustion. In order to compare stratification levels of different partially premixed combustion strategies or other combustion concepts, an objective and meaningful definition of “stratification level” is required. Such a definition is currently lacking; qualitative/quantitative definitions in the literature cannot properly distinguish various levels of stratification. The main purpose of this study is to objectively define combustion stratification (not to be confused with fuel stratification) based on high-speed OH* chemiluminescence imaging, which is assumed to provide spatial information regarding heat release. Stratification essentially being equivalent to spatial structure, we base our definition on two-dimensional Fourier transforms of photographs of OH* chemiluminescence. A light-duty optical diesel engine has been used to perform the OH* bandpass imaging on. Four experimental points are evaluated, with injection timings in the homogeneous regime as well as in the stratified partially premixed combustion regime. Two-dimensional Fourier transforms translate these chemiluminescence images into a range of spatial frequencies. The frequency information is used to define combustion stratification, using a novel normalization procedure. The results indicate that this new definition, based on Fourier analysis of OH* bandpass images, overcomes the drawbacks of previous definitions used in the literature and is a promising method to compare the level of combustion stratification between different experiments.