Bovine abortion is economically one of the most devastating problems faced by dairy farmers. Apart from non-infectious causes, several infectious pathogens are responsible for abortions, which sometimes manifests as abortion storms. Vaccine against several pathogens is available, in spite of that, abortions cause huge economic losses for the dairy sector. Timely and accurate identification of the etiological agent helps in adopting the mitigation steps to control the damage caused. In addition to the common abortion-causing pathogens such as Brucella abortus, Bovine herpesvirus-1 (BHV-1), bovine viral diarrhea virus (BVDV), several emerging viral causes are being investigated for their possible role in abortion, either exclusively or as co-infection. Molecular methods are widely accepted for the identification of the involved pathogens. However, these assays require individual screening against each pathogen which is time-consuming and uneconomical, hence the multiplex format of PCR assays has been adopted by several laboratories. Multiplexing in real-time PCR is a sensitive and reliable technique, but it requires trained manpower and sophisticated equipment which is largely unavailable in regional disease diagnostic laboratories in India. Hence, in this study, a user-friendly, ready-to-use, gel-based RT-PCR multiplex assay was developed for simultaneous detection of three common pathogens (B. abortus, BHV-1, and BVDV) and two emerging pathogens; bluetongue virus (BTV) as a cause of abortions in bovine and Schmallenberg virus (SBV). After the standardization of the assay, a panel of 211 samples was screened. A high degree of concordance was observed which indicates the developed multiplex PCR assay is reliable and has the potential for screening at regional diagnostic laboratories.