The compound (E,Z)-3-(4-hydroxy-3,5-dimethoxybenzylidene)indolin-2-one (indolinone) was identified from lipophilic woad extracts (Isatis tinctoria L., Brassicaceae) as a compound possessing potent histamine release inhibitory and anti-inflammatory properties . To further evaluate the potential of indolinone in terms of crossing the blood-brain barrier (BBB), we screened the compound in several in vitro cell-based human and animal BBB models. Therefore, we developed a quantitative LC-MS/MS method for the compound in modified Ringer HEPES buffer (RHB) and validated it according to FDA and EMA guidelines [2,3]. The calibration curve of indolinone in the range between 30.0 and 3000ng/ml was quadratic, and the limit of quantification was 30.0ng/ml. Dilution of samples up to 100-fold did not affect precision and accuracy. The carry-over was within acceptance criteria. Indolinone proved to be stable in RHB for 3h at room temperature (RT), and for three successive freeze/thaw cycles. The processed samples could be stored in the autosampler at 10°C for at least 28h. Moreover, indolinone was stable for at least 16 days in RHB when stored below -65°C. This validation study demonstrates that our method is specific, selective, precise, accurate, and capable to produce reliable results. In the immortalized human BBB mono-culture model, the apparent permeability coefficient from apical to basolateral (PappA→B), and the Papp from basolateral to apical (PappB→A) were 19.2±0.485×10(-6)cm/s and 21.7±0.326×10(-6)cm/s, respectively. For the primary rat/bovine BBB co-culture model a PappA→B of 27.1±1.67×10(-6)cm/s was determined. In the primary rat BBB triple co-culture model, the PappA→B and the PappB→A were 56.2±3.63×10(-6)cm/s and 34.6±1.41×10(-6)cm/s, respectively. The data obtained with the different models showed good correlation and were indicative of a high BBB permeation potential of indolinone confirmed by in silico prediction calculations. P-glycoprotein (P-gp) interaction for indolinone was studied with the aid of a calcein-AM uptake assay, and by calculation of the efflux ratio (ER) from the bidirectional permeability assays. For both bidirectional BBB models an ER below 2 was calculated, indicating that no active mediated transport mechanism is involved for indolinone. In porcine brain capillary endothelial cells (PBCECs), the calcein-AM uptake assay demonstrated that indolinone is neither a P-gp substrate nor a P-gp inhibitor and is accumulated into cells at high extent.