Cannabis products have been used in various fields of everyday life for many centuries, and applications in folk medicine and textile production have been well-known for many centuries. For traditional textile production, hemp fibers were extracted from the stems by water retting in stagnant or slow-moving waters. During this procedure, parts of the plant material‚ among them phytocannabinoids‚ are released into the water. Cannabinol (CBN) is an important degradation product of the predominant phytocannabinoids found in Cannabis species. Thus, it is an excellent indicator for present as well as ancient hemp water retting. In this study, we developed and validated a simple and fast method for the determination of CBN in sediment samples using high-performance thin-layer chromatography (HPTLC) combined with electrospray ionization mass spectrometry (ESI-MS), thereby testing different extraction and cleanup procedures‚ as well as various sorbents and solvents for planar chromatography. This method shows a satisfactory overall analytical performance with an average recovery rate of 73%. Our protocol enabled qualitative and quantitative analyses of CBN in samples of a bottom sediment core‚ having been obtained from a small lake in Northern India, where intense local retting of hemp was suggested in the past. The analyses showed a maximum CBN content in pollen zone 4 covering a depth range of 262–209 cm, dating from approximately 480 BCE to 1050 CE. These findings correlate with existing records of Cannabis-type pollen. Thus, the method we propose is a helpful tool to track ancient hemp retting activities.