Infection with Mycobacterium Tuberculosis represents a significant threat to people with immune disorders, such as HIV-positive individuals, and can result in significant health complications or death if not diagnosed and treated early. We present a centrifugal microfluidic platform for multiplexed detection of tuberculosis and HIV biomarkers in human whole blood with minimal sample preparation and a sample-to-answer time of 30 minutes. This multiplexed assay was developed for the detection of two M.tuberculosis secreted proteins, whose secretion represents an active and ongoing infection, as well as detection of HIV p24 protein and human anti-p24 antibodies. The limit of detection for this multiplex assay is in the pg/mL range for both HIV and M.tuberculosis proteins, making this assay potentially useful in the clinical diagnosis of both HIV and Tuberculosis proteins indicative of active infection. Antigen detection for the HIV assay sensitivity was 89%, the specificity 85%. Serological detection had 100% sensitivity and specificity for the limited sample pool. The centrifugal microfluidic platform presented here offers the potential for a portable, fast and inexpensive multiplexed diagnostic device that can be used in resource-limited settings for diagnosis of TB and HIV.