The thin-layer chromatography-flame ionization detection (TLC-FID) Iatroscan system is a technique which is still being evolved. Quantification with the TLC-FID system relies heavily on the accurate setting up and calibration of the instrument. An appreciation of the factors that influence the analysis can eliminate significant errors. At least a few of the numerous operating variables need to be fixed to obtain coherent results from different laboratories. Hydrogenation of the sample is recommended in order to improve quantification with the Iatroscan system. The improved reproducibility obtained with automatic sample spotters compared with manual spotting indicates that autosampling is highly advisable.