in current treatment plans of intensity-modulated proton therapy, high-energy beams are usually assigned larger weights than low-energy beams. Using this form of beam delivery strategy cannot effectively use the biological advantages of low-energy and high-linear energy transfer (LET) protons present within the Bragg peak. However, the planning optimizer can be adjusted to alter the intensity of each beamlet, thus maintaining an identical target dose while increasing the weights of low-energy beams to elevate the Let therein. the objective of this study was to experimentally validate the enhanced biological effects using a novel beam delivery strategy with elevated LET. We used Monte Carlo and optimization algorithms to generate two different intensity-modulation patterns, namely to form a downslope and a flat dose field in the target. We spatially mapped the biological effects using high-content automated assays by employing an upgraded biophysical system with improved accuracy and precision of collected data. In vitro results in cancer cells show that using two opposed downslope fields results in a more biologically effective dose, which may have the clinical potential to increase the therapeutic index of proton therapy.Worldwide the number of proton therapy centers has increased dramatically in recent years 1 . The expansion of proton therapy centers can be attributed to multiple factors. The first and foremost advantage of protons is that a Bragg-peak dose profile appears at the end of the range of a proton beam. The use of different beam modulation and shaping techniques makes it possible to deliver a uniform high dose to the tumors while sparing the surrounding normal tissues 2 . The uniform target dose can be achieved using the passive-scattering proton therapy (PSPT) technique or the more advanced intensity-modulated proton therapy (IMPT) technique employing scanned beams. Importantly, some clinical trials have indicated the advantages of proton therapy over traditional photon-based therapy 3-6 . However, there are still many challenging issues in modern proton therapy. One of the them is that the radiobiological characteristics of proton therapy have yet to be completely understood. In current clinical practice, the relative biological effectiveness (RBE) of protons to reference photons, such as x-rays from a