Direct delivery of aerosol or vapor to the apical surface of cells (ALI) allows more relevant exposure for in vitro toxicological evaluation of inhalable chemicals. In this study, we quantitatively characterized the aerosol delivery in a commercially available ALI in vitro exposure system (VITROCELL^® VC1/7 puffing machine and Vitrocell^® Ames 48 [Ames 48]) using a prototype e-vapor product (MarkTen^® e-cigarette with a prototype e-liquid containing propylene glycol, glycerin, nicotine, and water). The e-vapor product, with a fully-charged battery, was puffed using a 55 ml puff over 5 seconds, with a 30 second inter puff period, by a VC1/7 puffing machine. As specified by the manufacturer, e-vapor aerosol was pulled into the VC1 puffing machine and then pushed into the exposure system over 8 seconds. Aerosol mass was collected and measured gravimetrically following the first 20 puffs at the exit of each puffing unit (seven VC1s) and the inlet and outlet of the AMES 48. The average aerosol mass delivery (calculated as measured mass/total product weight loss × 100 %) was 68.6 %, 49.1 %, and 46.6 %, respectively, with about 0.48 %-0.66 % of aerosol mass delivered to the exposure inserts. Results suggested about 50 % aerosol loss in the aerosol transportation path (VC1 and tubing) prior to entry into the exposure system. To minimize the aerosol loss and consequently increase the aerosol delivery to the inserts, we revised the aerosol delivery method by shortening the aerosol transportation path. With the revised puffing method, the estimated aerosol delivery at the inlet of the in vitro exposure system was about 93.5 %-95.3 %, with increased aerosol delivery to 1.0 %-1.2 % in the exposure inserts.