This study aimed to evaluate and compare serum drug concentrations and plasma free-VEGF concentrations in patients with AMD, DME, or RVO receiving intravitreal injections of aflibercept, bevacizumab or ranibizumab. This prospective study enrolled patients from several offices of a single private practice who were naive to anti-VEGF therapy or had not received treatment with anti-VEGF for at least four months. Patients were sorted into three groups depending on disease aetiology and given three monthly injections. Blood samples were collected at screening, three hours after injection, and at days one, three, seven and 28 after Dose 1 and Dose 3 for PK (population pharmacokinetic) and systemic VEGF analyses. A total of 151 patients were enrolled in this study, 57 in the AMD group, 46 in the DME group and 48 in the RVO group. Mean ± SD serum half-lives after intravitreal administration for aflibercept, bevacizumab, and ranibizumab were 11.4 ± 4.8 days (n=39), 18.7 ± 5.8 days (n=7), and 5.8 ± 1.8 days (n=43), respectively. Systemic exposure for each anti-VEGF therapeutic did not seem to differ by indication and was consistently highest with bevacizumab, followed by aflibercept, and lowest with ranibizumab. For Dose 1 and Dose 3, the greatest decreases in plasma free-VEGF levels were observed with aflibercept for all three indications. This study provides evidence that aflibercept, bevacizumab, and ranibizumab exhibit different systemic exposures and effects on systemic VEGF after intravitreal injection.