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Two new HPTLC methods for quantification of isopropyl-9H-thioxanthen-9-one (ITX) in milk, yoghurt and fat samples have been developed. Extraction of ITX from milk and yoghurt was performed with a mixture of cyclohexane and ethyl acetate by employment of accelerated solvent extraction (ASE). For soy bean oil and margarine, a simple partitioning of ITX into acetonitrile was used. ITX and 2,4-diethyl-9H-thioxanthen-9-one (DTX) used as internal standard have been separated on silica gel 60 HPTLC plates with a mixture of toluene and n-hexane (4:1, v/v) and on RP18 HPTLC plates with a mixture of acetonitrile and water (9:1, v/v). Development was performed anti-parallel from both plate sides leading to a throughput of 36 separations in 7 min. Fluorescence measurement at 254/>400 nm was used for quantification. Limits of detection (S/N of 3) have been established to be 64 pg for ITX and DTX on both types of HPTLC plates. In fatty matrix (spiked butter) LOD of ITX was determined to be 1 μg kg−1. In the working range monitored (20–200 μg kg−1) polynomial regression of ITX showed a relative standard deviation (sdv) of ±1.51 % ( r=0.99981). Starting with the limit of quantification the response was linear (sdv=±2.18 %, r=0.99893). Regarding repeatability ( n=9) a coefficient of variation (CV) of 1.1 % was obtained for ITX at 32 ng on silica gel plates and of 2.9 % on reversed-phase plates. Repeatabilities ( n=4) of ITX determination at 20, 50 and 100 μg kg−1 in milk, yoghurt, soybean oil and margarine showed CVs between ±1.0 and 6.4 %. The results prove that modern planar chromatography is a rapid and cost-efficient alternative method to quantify ITX in milk-based or fatty matrices. Only positive results are confirmed by online ESI/MS in the SIM mode (LOQ 128 pg) and by DART/MS involving a minimal employment of the MS device, which is a further advantage of HPTLC. Overall mean recovery rates of ITX at 20 or 50 and 100 μg kg−1 ( n=8) were 41 % for milk, 70 % for yoghurt, 6 % for margarine and 12 % for soy bean oil. However, with the internal standard correction recoveries were about 130 % for milk and yoghurt and 70 and 97 % for margarine and soy bean oil, respectively. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]