Adaptive optics flood illumination ophthalmoscopy (AO-FIO) allows imaging of the cone photoreceptor in the living human retina. However, clinical interpretation of the AO-FIO image remains challenging due to suboptimal quality arising from residual uncorrected wavefront aberrations and rapid eye motion. An objective method of assessing image quality is necessary to determine whether an AO-FIO image is suitable for grading and diagnostic purpose. In this work, we explore the use of focus measure operators as a surrogate measure of AO-FIO image quality. A set of operators are tested on data sets acquired at different focal depths and different retinal locations from healthy volunteers. Our results demonstrate differences in focus measure operator performance in quantifying AO-FIO image quality. Further, we discuss the potential application of the selected focus operators in (i) selection of the best quality AO-FIO image from a series of images collected at the same retinal location and (ii) assessment of longitudinal changes in the diseased retina. Focus function could be incorporated into real-time AO-FIO image processing and provide an initial automated quality assessment during image acquisition or reading center grading.