(1) The objective of the present study was to identify suitable parameters to determine the (degree of) freshness of Bell pepper fruit of three colors (yellow, red, and green) over a two-week period including the occurrence of shrivel using non-destructive real-time measurements (2) Materials and methods: Surface glossiness was measured non-destructively with a luster sensor type CZ-H72 (Keyence Co., Osaka, Japan), a colorimeter, a spectrometer and a profilometer type VR-5200 (Keyence) to obtain RGB images. (3) Results: During storage and shelf life, bell pepper fruit of initially 230–245 g lost 2.9–4.8 g FW per day at 17 °C and 55% rh. Shriveling started at 6–8% weight loss after 4–5 days and became more pronounced. Glossiness decreased from 450–500 a.u. with fresh fruit without shrivel, 280–310 a.u. with moderately shriveled fruit to 80–90 a.u. with severely shriveled fruit irrespective of color against a background of extless40 a.u. within the same color, e.g., light red and dark red. Non-invasive color measurements showed no decline in Lab values (chlorophyll content), irrespective of fruit color and degree of shrivel. RGB images, converted into false color images, showed a concomitant increase in surface roughness (Sa) from Sa = ca. 2 µm for fresh and glossy, Sa = ca. 7 µm for moderately shriveled to Sa = ca. 24 µm for severely shriveled rough surfaces of stored pepper fruit, equivalent to a 12-fold increase in surface roughness. The light reflectance peak at 630–633 nm was universal, irrespective of fruit color and freshness. Hence, a freshness index based on (a) luster values ≥ 450 a.u., (b) Sa ≤ 2 µm and (c) the difference in relative reflectance in % between 630 nm and 500 nm is suggested. The latter values declined from ca. 40% for fresh red Bell pepper, ca. 32% after 6 days when shriveling had started, to ca. 21% after 12 days, but varied with fruit color. (4) Conclusion: overall, it can be concluded that color measurements were unsuitable to determine the freshness of Bell pepper fruit, whereas profilometer, luster sensor, and light reflectance spectra were suitable candidates as a novel opto-electronic approach for defining and parametrizing fruit freshness.