A prediction of the standard ΛCDM cosmological model, also confirmed by N-body simulations, is that dark matter (DM) halos are teeming with numerous self-bound substructure, or subhalos. The precise properties of these subhalos represent important probes of the underlying cosmological model. In this work, we use data from the N-body Via Lactea II and ELVIS Milky Way-size simulations to learn about the structure of subhalos with masses 10 6 − 10 11 h −1 M . Thanks to a superb subhalo statistics, by taking a profileindependent approach, we study subhalo properties as a function of the distance to the host halo center and subhalo mass, and provide a set of fits that, including both dependences, accurately describe the subhalo structure. With this at hand, we also investigate the role of subhalos on the search for DM via its annihilation products. Indeed, previous work has shown that subhalos are expected to boost the DM signal of their host halos significantly. Yet, these works have traditionally assumed that subhalos exhibit similar structural properties than those of field halos of the same mass, while it is well known from simulations that subhalos are more concentrated. Building upon the results from our N-body data analysis, we refine the substructure boost model of Sánchez-Conde & Prada (2014). We find boost values that are a factor 2 − 3 higher than previous ones. We further refine our boost model to include unavoidable tidal stripping effects on the subhalo population. For field halos, this only introduces a moderate (∼ 20% − 30%) suppression of the boost. Yet, for subhalos like those hosting the dwarf satellite galaxies of the Milky Way, tidal stripping does play a critical role, the total boost for these objects being only at the level of a few tens of percent in the most optimistic cases. Finally, we provide a parametrization of the boost factor for field halos that can be safely applied over a wide halo mass range.