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Purpose: To compare the use of toluidine blue 1% eye drops with anterior segment optical coherence tomography (OCT) for the determination of tumour margins in patients with ocular surface squamous neoplasia (OSSN)., Methods: The study was conducted from July 2020 to June 2021 at the Ocular Oncology department at the Federal University of São Paulo, Brazil. Slit-lamp photographs after toluidine blue staining and OCT of the anterior segment were taken on the same day from patients with OSSN. Photographs and OCT images were analyzed quantitatively using the software ImageJ and IMAGEnet®, respectively. The agreement between techniques was evaluated qualitatively through the Bland-Altman graph and quantitatively through intraclass correlation (ICC)., Results: A total of 21 participants (71.43% males) with a clinical diagnosis of OSSN were included in the study. The average + SD diameter along the chosen axes was 4.43 ± 2.08 mm with OCT of 4.37 ± 2.03 mm with toluidine blue, a difference not statistically significant (p = 0.2891). The Bland-Altman analysis indicated a good qualitative agreement between the methods, with all cases inserted within the limits of agreement from -0.3217 to 0.4268. The ICC quantitative analysis showed an almost perfect agreement of 99.57% (95%CI: 98.96-99.83%; p < 0.001)., Conclusions: Our findings showed that OCT and toluidine eye drops are equivalent in determining margins for tumour measurements, which is particularly relevant in low-income settings where anterior segment OCT is not available. The use of toluidine blue 1% could be an useful alternative to quantify the size of the tumour, help to monitor tumour growth, and outline margins for surgical planning., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.