A high density of Demodex infestation in human eyelids and eyelash follicles has been implicated in a variety of ocular surface conditions. However, Demodex infestation often goes undiagnosed and untreated, due to an overlap in signs and symptoms with other sources of ocular surface inflammation. We sought to refine the diagnostic technique for outpatient assessment for Demodex infestation and determine prevalence of Demodex mites in the hair follicles of eyelashes from patients in a standard ophthalmic practice. Patients and Methods: Patients recruited from a single outpatient ophthalmology clinic were examined for the presence of Demodex mites following standard ophthalmic consultation. During anterior segment biomicroscopic examination, investigators searched for cylindrical dandruff and collarettes around the base of patient eyelashes. These were removed, and individual eyelashes manipulated with tweezers to reveal Demodex mites. Presence of Demodex, cylindrical dandruff and collarettes, time taken to identify the first Demodex mite, mean number of Demodex mites per hair shaft, and patient-reported symptoms of blepharitis were recorded and analyzed to determine potential correlations. Results: A total of 173 patients were recruited, of whom 106 were included in Demodex prevalence analyses. Demodex infestation was identified in 100/106 (94%) patients, with a mean time of 2 minutes and 20 seconds taken to find the first Demodex mite and a mean number of 3 mites identified per hair shaft. Presence of Demodex significantly correlated with the presence of cylindrical dandruff (98/106 [92%] patients; p < 0.001), but not with the presence of collarettes (62/106 [58%] patients; p = 0.230) or symptoms of blepharitis (15/106 [14%] patients; p = 0.591). Conclusion: These results highlight the pervasiveness of Demodex infestation, predicted by the presence of cylindrical dandruff, in the eyelashes of patients in a standard ophthalmic practice. They support the use of a non-invasive diagnostic technique to allow fast, simple identification of Demodex by ophthalmologists and optometrists in outpatient clinical practice.