To quantitatively access penumbra sharpening and scattering by adaptive aperture (AA) under various beam conditions and clinical cases for a Mevion S250i compact pencil beam scanning proton therapy system. Methods: First, in-air measurements were performed using a scintillation detector for single spot profile and lateral penumbra for five square field sizes (3 9 3 to 18 9 18 cm 2 ), three energies (33.04, 147.36, and 227.16 MeV), and three snout positions (5, 15, and 33.6 cm) with Open and AA field. Second, treatment plans were generated in RayStation treatment planning system (TPS) for various combination of target size (3-and 10-cm cube), target depth (5, 10, and 15 cm) and air gap (5-20 cm) for both Open and AA field. These plans were delivered to EDR2 films in the solid water and penumbra reduction by AA was quantified. Third, the effect of the AA scattered protons on the surface dose was studied at 5 mm depth by EDR2 film and the RayStation TPS computation. Finally, dosimetric advantage of AA over Open field was studied for five brain and five prostate cases using the TPS simulation. Results: The spot size changed dramatically from 3.8 mm at proton beam energy of 227.15 MeV to 29.4 mm at energy 33.04 MeV. In-air measurements showed that AA substantially reduced the lateral penumbra by 30% to 60%. The EDR2 film measurements in solid water presented the maximum penumbra reduction of 10 to 14 mm depending on the target size. The maximum increase of 25% in field edge dose at 5 mm depth as compared to central axis was observed. The substantial penumbra reduction by AA produced less dose to critical structures for all the prostate and brain cases. Conclusions: Adaptive aperture sharpens the penumbra by factor of two to three depending upon the beam condition. The absolute penumbra reduction with AA was more noticeable for shallower target, smaller target, and larger air gap. The AA-scattered protons contributed to increase in surface dose. Clinically, AA reduced the doses to critical structures.