Background & Aim: This study aims to evaluate the prevalence of undiagnosed hepatitis delta in southern Spain (Andalusia) and assess the effectiveness and cost-efficiency of implementing reflex testing for hepatitis D detection in HBsAg-positive patients. Patient & Methods: A multicenter ambispective study was conducted in 17 hospitals in Andalusia. The retrospective phase analyzed diagnostic processes for hepatitis delta from January 2018 to June 2022, focusing on HBsAg-positive patients. The prospective phase, from October 2022 to March 2023, implemented reflex testing, performing anti-HDV serology on all HBsAg-positive patients without prior testing. HDV RNA testing was conducted on those who tested positive for anti-HDV. Results: In the retrospective phase, out of 18,583 HBsAg-positive patients, anti-HDV tests were performed on 3,436 (18%), identifying 205 (6%) positive cases. HDV RNA was tested in 158 (77%) anti-HDV-positive patients, with 69 (44%) testing positive. In the prospective phase, out of 2,384 HBsAg-positive patients without prior anti-HDV testing, 2,293 (96%) were tested, identifying 109 (4.7%) positive cases. HDV RNA was analyzed in 97 (89%) anti-HDV-positive patients, with 30 (31%) testing positive. Reflex testing increased anti-HDV detection by 77%, resulting in a fourfold increase in detecting anti-HDV-positive patients and a threefold increase in detecting HDV RNA-positive patients, reducing undiagnosed HDV RNA-positive cases to 4% compared to 45% with clinical practice. Cost analysis indicated a saving of €265,954 with reflex testing. Discussion: Reflex testing significantly improves HDV detection and reduces healthcare costs. It simplifies the diagnostic process, increasing the detection rate of hidden chronic hepatitis delta patients and proving to be an efficient strategy for managing chronic hepatitis B patients.