Mushroom tyrosinase has long been used to search its own inhibitor in the cosmetic industry, since this enzyme is easy to obtain and show high enzyme activity. However its structure and catalytic nature are different from those of mouse and human cell lines. In order to provide a correct and convenient tool for screening inhibitors of tyrosinase, tyrosinases from mouse and human melanocytes were obtained from cell culture. Km for L-DOPA as a substrate of mushroom, mouse and human tyrosinases were 0.36 mM, 0.47 mM and 0.42 mM, respectively. Tyrosinase-inhibition patterns by arbutin were competitive in mushroom, noncompetitive in mouse and human. Tyrosinase-inhibition patterns by kojic acid showed mixed type in mushroom, competitive type in mouse, and partial mixed type in human. IC50 of arbutin for mushroom, mouse and human tyrosinases were 85.1 mM, 1.1 mM and 5.8 mM, respectively. IC50 of kojic acid were 7.4 μM, 57.8 μM and 146.2 μM, respectively. Data show that IC50 of arbutin for mouse and human tyrosinases were much lower than that for mushroom tyrosinase, and IC50 of kojic acid for mouse and human tyrosinases were much higher than that for mushroom tyrosinase. Therefore, mushroom tyrosinase is not suitable for the screening and evaluation of tyrosinase inhibitors. Spearman correlation analysis was performed on the test methods used for the whitening screening and evaluation. Correlation coefficients between mushroom tyrosinase and antioxidant test were 0.351 and 0.397 in ABTS and DPPH, respectively. However, there was no correlation with melanin production. Through the mouse tyrosinase test, the whitening efficacy of the Drynariae fortunei Rhizoma(DF), which was not previously reported, was confirmed. The DF extract showed excellent whitening activity of 84% at 100 ug/ml of CHCl3 fraction. The DFC3 and DFC4 column fractions were 52 and 58% at 100 ug/ml, respectively, and the final purified DFC34H-3 fraction was 58% at 25 ug/ml. However, the DFC34H-3 fraction showed cytotoxicity with a cell proliferation rate of 62% at 20 ug/ml. Therefore, in order to utilize DF as a whitening agent, it is considered that it is suitable to use as a solvent fraction. The molecular weight of DFC34H-3 was estimated at 256 from the molecular ion peak m/z 255 [M-H]- in the negative FAB-MS and m/z 289 [M+Na]+ in positive FAB-MS. The results of 1H-NMR and 13C-NMR measurements of DFC34H-3 were inconsistent with previously reported compounds in DF. It is considered to be a compound not reported in DF.