Many mobile phases, including solvent systems with one and two polar modifiers as well as some multiple development techniques and gradient elutions, were examined to select the best thin-layer chromatographic conditions on silica gel F254 plates for micropreparative isolation of 10-deacetylbaccatin III (10-DAB III), baccatin III, paclitaxel, and cephalomannine from methanolic extracts obtained from fresh and dried needles and stems of Taxus baccata L. In case of each solvent system and development technique, R F , k, and α values of 10-DAB III and paclitaxel were determined as well as the separation of compounds coextracted with taxoids, and the whole yew extract. The best mobile phase, benzene-chloroform-acetone-methanol (20:92.5:15:7.5) used twice over a distance of 15 cm was applied for isolation of analyzed taxoids by preparative TLC in connection with RP-HPLC, (C-18) quantitative determination using two mobile phases (30% acetonitrile in water to measure of 10-DAB III levels and 50% acetonitrile in water for baccatin III, paclitaxel, and cephalomannine). In this way, concentrations of 10-DAB III, baccatin III, paclitaxel, and cephalomannine were determined in the fresh needles and stems of Taxus baccata L. collected during the late autumn-spring period (November to April) as well as in dried plant material. Observed retention behavior of baccatin III on silica gel in comparison with its precursor (10-DAB III) is discussed. Some of the investigated solvent systems can be applied for improved column chromatographic separation of the analyzed taxoids.