The programmed multiple development thin-layer chromatography (PMD-TLC) technique was compared with the conventional chromatographic technique on both normal and HPTLC plates. The potency of this high-performance TLC is illustrated and discussed by means of the data obtained with the determination of digitoxin in human serum. Improvement of the efficiency of TLC, resulting in a better resolution and sensitivity with PMD-TLC using using HPTLC plates, makes this high-performance TLC technique comparable to high-performance liquid chromatography. This TLC approach might lead to a sensitive, rapid, selective and simply assay for routine serial analyses and, because of its specificity and flexibility, it may facilitate drug interaction studies.