LC-MS/MS method was used to determine if BACE1 or γ-secretase inhibitor were permeable into neuro-spheroids. Spheroids were collected after 2 days of treatment and subjected to LC-MS/MS quantification. The LC-MS/MS system consists of UltiMate 3000 UHPLC automated system coupled with TSQ Quantiva triple quadrupole Mass Spectrometer (Thermo Fisher, Waltham, MA). Samples were prepared by adding 250ul ice cold acetonitrile to each sample vial containing pre-washed 3D neuron cells. Then, each sample was sonicated and vortexed vigorously while keeping the sample cold by immersion into ice between the steps. These steps were repeated until all cells were disrupted. After samples were centrifuged at 12,000 ×g for 10min, the supernatant was aliquoted and diluted with mobile phase A, and then transferred into a HPLC vial for LC-MS/MS analysis. The chromatographic separation was performed on a Kinetex C18 column (50 x 2.1mm, 2.6 um particle size, Phenomenex, Torrance, CA) with mobile phase consisting of water with 0.1% formic acid (mobile phase A) and acetonitrile with 0.1% formic acid (mobile phase B), running a linear gradient from 1 to 90% for 13 min, and
