Sugars, in particular glucose and fructose, are the main constituents of honey, comprising up to 85% of its total weight. A high-performance thin-layer chromatography (HPTLC) method to identify and quantify common sugars (glucose, fructose and sucrose) in honey has been developed and fully validated according to the International Conference on Harmonisation guidelines. It allows to determine a honey’s fructose-to-glucose ratio, which is not only an important authentication and quality control parameter, but also an indication of its tendency to crystallise. The HPTLC analysis is easy to perform, accurate, precise, specific and sensitive and requires only minimal sample preparation. With a limit of detection/limit of quantification of 21.98 ng/66.62 ng for fructose, 33.00 ng/100.00 ng for glucose and 21.17 ng/64.15 ng for sucrose, the sensitivity of the method has been greatly improved compared to other HPTLC-based approaches. An additional advantage is the method’s simplicity and fast processing time as it only requires a single development step without plate or sample pre-treatment.