Background/Objectives: The creation of clinically relevant in vitro white spot lesions (WSLs) is essential for the reliable evaluation of diagnostic devices and remineralization therapies. This study aims to optimize the in vitro WSL creation protocol across various demineralization periods to produce lesions comparable to post-orthodontic WSLs, using different diagnostic methods. Methods: Forty extracted upper first premolars were randomly allocated into four groups (ten per group) and exposed to hydroxyethylcellulose (HEC) gel for demineralization periods of 7, 10, 14, and 21 days. Lesions were evaluated by ICDAS II, DIAGNOdent pen, and a Scanning Electron Microscope (SEM). The correlations among the diagnostic methods were analyzed. Results: According to ICDAS II scoring, a score of 1 was predominant in the 7-day group, while the others exhibited a score of 2. DIAGNOdent readings increased significantly with longer durations (G7 mean: 7.80 ± 2.04; G21 mean: 82.70 ± 13.11). SEM analysis indicated an increase in lesion depth with extended exposure durations (G7 mean: 46.59 ± 2.72; G21 mean: 101.7 ± 9.75). Nevertheless, the rate of increase in lesion depth diminished with prolonged exposure. A strong correlation was observed between lesion depth and DIAGNOdent readings (r = 0.90). Additionally, ICDAS II demonstrated moderate correlations with DIAGNOdent and lesion depth (r = 0.69 and 0.66, respectively). Conclusions: A 10-day demineralization period was identified as the most favorable and reproducible, producing lesions with an intact surface and consistent with DIAGNOdent criteria. This duration may serve as the optimal model for evaluating intensive remineralization therapies and diagnostic methods prior to clinical implementation.
