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To evaluate the efficacy of a prophylactic regimen of daily topical 0.5% moxifloxacin and 5% povidone–iodine (PI) in patients with Boston type I keratoprosthesis (KPro) and to assess the applicability of a novel molecular diagnostic technique to analyze the ocular surface microbiota in these patients.Ten patients had their inferior conjunctival fornix sampled for standard culture methods before the addition of topical 5% PI to the prophylactic regimen and were considered the control group (group 1). The inferior conjunctival fornix and the KPro–donor cornea interface of 10 patients treated with the mentioned prophylactic regimen were sampled and analyzed by standard culture methods and using a polymerase chain reaction/electrospray ionization mass spectrometry assay (group 2).Samples from the inferior conjunctival fornix were positive for coagulase-negative staphylococcus in 3 patients and for Aerobasidium pullulans in 1 patient in group 1. The inferior conjunctival fornix and the KPro–donor cornea interface scrapings were positive for coagulase-negative staphylococcus in 2 patients and 1 patient, respectively, in group 2. No bacteria and fungi growth were detected in any patient from group 2 with the molecular diagnostic approach. None of the patients with culture-positive results developed keratitis or endophthalmitis during the study.Topical 0.5% moxifloxacin associated with topical 5% PI is an effective prophylactic regimen in patients with Boston type I KPro. The molecular diagnostic approach using serial polymerase chain reaction and mass spectrometry was comparable with standard microbiologic techniques as a surveillance tool in these patients.