In this contribution, we examine the photophysical properties of 15 totally trans–trans 1,4-distyrylbenzene derivatives (DSBs) functionalized with different electron-donating (ED) and electron-withdrawing (EW) groups by experimental and computational methodologies. We use UV–vis and fluorescence spectroscopies to determine the experimental optical properties such as the maximum absorption (λabsexp) and emission (λemexp) wavelengths, the highest occupied molecular orbital–lowest unoccupied molecular orbital (HOMO–LUMO) energy gaps (ΔEabsexp), the molar extinction coefficients (ε), the fluorescence quantum yields (Φf), and the fluorescence lifetimes (τ). We also calculate the experimental spontaneous emission decay rate (krexp) and correlate all of these magnitudes to the corresponding calculated properties, maximum absorption (λabscal) and emission (λemcal) wavelengths, vertical transition energies (ΔEabscal), oscillator strength (Fosc), and spontaneous emission decay rate (krcal), obtained by the time-dependent density functional theory method. We analyze the effect of the electronic nature of the substituents on the properties of the DSBs, finding that the ED and EW groups lead to bathochromic shifts. This is consistent with the decrease of ΔE values as the strength of ED and EW substituents increases. We find excellent correlations between calculated and experimental values for λabs, λem, and ΔEabs (r ∼ 0.99–0.95). Additionally, the correlations between the relative ε with Fosc values and the kr values are in good agreement (r ∼ 0.88–0.72) with the experimental properties. Overall, we find that for substituted 1,4-DSBs, computational chemistry is an excellent tool to predict structure–property relationships, which can be useful to forecast the properties of their polymeric analogues, which are usually difficult to determine experimentally.