Thin-layer chromatography (TLC) has been in use for more than the past 40 years. In the field of analytical chemistry, TLC is considered to be the most simple, robust, economical, and rapid technique. TLC is considered to be an ideal chromatographic system for clinical laboratories. The adsorbents used as stationary phases in TLC techniques can be polar or nonpolar. The slurry is spread on the TLC plate by use of a manual or mechanical spreading device. The air-dried TLC plates are activated in a hot air oven at about 110 to 120°C for 1 to 2 h. The validation of TLC methods requires the demonstration of calibration linearity, specificity, sensitivity, extraction recovery, precision, and accuracy. The relative simplicity of the method and inexpensive cost of operation make it the method of choice in clinical chemistry. Proper storage and preservation of the sample is also important in clinical chemistry. TLC has also gained popularity for routine examination of porphyrins in clinical chemistry.