Background One of the most unfavorable side effects of fixed orthodontic treatment is white spot lesions (WSLs). Although the most important approach is prevention of WSLs, it is also essential to evaluate the efficacy of the remineralization agents. However, there is no concurrence in the literature with respect to the remineralization process of these agents. The objective of the present study was to evaluate the effects of different fluoride varnishes, enamel matrix protein, and self-assembling peptide derivatives with varying chemical compositions on remineralization of artificially created WSLs in vitro using quantitative light-induced fluorescence (QLF). Methods Artificial WSLs were created on bovine enamel samples using acidic buffer solution (pH 5, 10 days). Specimens were randomly allocated to six groups (n = 10/group): (1) Emdogain (Straumann, Basel, Switzerland), (2) Curodont Repair (Credentis AG, Switzerland), (3) Duraphat (Colgate-Palmolive, New York, NY), (4) Clinpro XT (3 M ESPE, Pymble, New South Wales, Australia), (5) Enamel Pro Varnish (Premier Dental Products, PA, USA), and (6) control (untreated). The agents were applied to the WSLs according to the manufacturers' instructions. Fluorescence loss (Delta F), lesion area (area), and impact (Delta Q) values of enamel surfaces were quantified by QLF-D Biluminator(TM) (Inspektor-Pro, Amsterdam, The Netherlands) at baseline and after 7, 14, and 21 days of application of the respective materials. Results Delta F value presented a significantly decreasing trend throughout the 21 days for all groups except the Duraphat and Enamel Pro varnishes. The changes between 14th and 21st days of the Clinpro XT varnish application were significantly higher than Emdogain, Curodont, and Enamel Pro. The Curodont group showed higher lesion area changes between the first and second week in comparison to the Emdogain, Clinpro XT, and Enamel Pro groups, whereas Clinpro XT assured the highest reduction from the second to the third week of the observation period. Conclusions The fluorescence loss was significantly reduced with enamel matrix protein, self-assembling peptide, and light-curable fluoride varnishes in the analysis for 21 days. Curodont and Clinpro XT were effective in diminishing the fluorescence loss and lesion area compared to the Duraphat, Enamel Pro fluoride varnishes, and Emdogain in different time points.