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Background: Visceral leishmaniasis (VL) is diagnosed by microscopic confirmation of the parasite in bone marrow, spleen or lymph node aspirates. These procedures are unsuitable for rapid diagnosis of VL in field settings. The development of rK39-based rapid diagnostic tests (RDT) revolutionized diagnosis of VL by offering high sensitivity and specificity in detecting disease in the Indian subcontinent; however, these tests have been less reliable in the African subcontinent (sensitivity range of 75–85%, specificity of 70–92%). We have addressed limitations of the rK39 with a new synthetic polyprotein, rK28, followed by development and evaluation of two new rK28-based RDT prototype platforms. Methodology/Principal Findings: Evaluation of 62 VL-confirmed sera from Sudan provided sensitivities of 96.8% and 93.6% (95% CI = K28: 88.83–99.61%; K39: 84.30–98.21%) and specificities of 96.2% and 92.4% (95% CI = K28: 90.53–98.95%; K39: 85.54–96.65%) for rK28 and rK39, respectively. Of greater interest was the observation that individual VL sera with low rK39 reactivity often had much higher rK28 reactivity. This characteristic of the fusion protein was exploited in the development of rK28 rapid tests, which may prove to be crucial in detecting VL among patients with low rK39 antibody levels. Evaluation of two prototype lateral flow-based rK28 rapid tests on 53 VL patients in Sudan and 73 VL patients in Bangladesh provided promisingly high sensitivities (95.9% [95% CI = 88.46–99.1 in Sudan and 98.1% [95% CI = 89.93–99.95%] in Bangladesh) compared to the rK39 RDT (sensitivities of 86.3% [95% CI = 76.25–93.23%] in Sudan and 88.7% [95% CI = 76.97–95.73%] in Bangladesh). Conclusions/Significance: Our study compares the diagnostic accuracy of rK39 and rK28 in detecting active VL cases and our findings indicate that rK28 polyprotein has great potential as a serodiagnostic tool. A new rK28-based RDT will prove to be a valuable asset in simplifying VL disease confirmation at the point-of-care. Author Summary: Visceral Leishmaniasis caused by Leishmania donovani is endemic in several parts of South Asia, East Africa, South and Central America. It is a vector-borne disease transmitted by bites of infected sand flies and often fatal in the absence of chemotherapy. Timely diagnosis is an essential first step in providing proper patient care and in controlling transmission. VL diagnosis in East Africa and Latin America are currently based on microscopic confirmation of parasites in tissue aspirates. The Kalazar Detect rapid test is widely used as a confirmatory test in India with very high accuracy, but sensitivity issues have severely limited its usefulness in the African sub-continent. Direct Agglutination Test is another confirmatory test used widely in East Africa and offers high sensitivity but is not field-friendly. We report on the design of a novel synthetic fusion protein capable of sequestering antibodies against three different Leishmania donovani antigens and the development of point-of-care tests for improving VL diagnosis. We believe the ease of use of these rapid tests and their high accuracy in detecting VL cases could make them useful as a first-line test, thereby eliminating the need for painful biopsies and ensuring better patient care. [ABSTRACT FROM AUTHOR]