Development of next-generation fluorescent probes is a key element in the quest for a greater understanding of complex biological environments (e.g., membranes) by bioimaging. Such fluorescence-based techniques rely on specialized small molecules that possess excellent fluorescent properties but also do not perturb the native biological environment in which they reside. Herein we present a theoretical/computational strategy for the design of novel optical probes for sensing in membranes based on the parent chromophore Nile Red. Using a combination of time-dependent density functional theory (TD-DFT) and molecular dynamics (MD), we have studied the optical properties and accommodation in a model membrane of Nile Red and eight analogs. Special attention has been given to the design of probes with improved solvatochromism and two-photon absorption (2PA) without altering the membrane properties. Of the eight studied analogs, two probes were found to possess attractive probe features and are hence suggested to be taken forward to chemical synthesis and experimental exploration.