For five of the last ten years tuberculosis (TB) disease, caused by active infection with Mycobacterium tuberculosis (M.tb), has been the leading cause of death globally. Therapies designed to complement drug treatment or disrupt M.tb transmission are urgently needed while next generation vaccines and drugs are being evaluated. Mycobacteriophages are examples of readily abundant biologics that are bactericidal against M.tb and can help fill this immediate gap. However, it has been reported that repeated intravenous (i.v.) phage treatment for other mycobacterial infections can induce a host humoral immune response that may interfere with therapy. Here we aimed to develop a total IgG, IgM and IgA high-throughput ELISA to evaluate the magnitude of anti-phage antibody responses in a preclinical C57BL/6 therapeutic mouse model. We selected representative anti-M.tb phages Muddy HRMN0157-2 (Muddy) and FionnbharthΔ45Δ47 (Fionnbharth), which have been shown to have lytic activity against M.tb. Cohorts of mice were given 10^8 pfu/mL of phage in 200µl by i.v. weekly for four weeks and serum was collected weekly for five weeks. To measure serum antibody levels, 384 well ELISA plates were coated with 10^8 pfu/well of phage in either traditional coating buffer or phage buffer and incubated overnight at 4°C. Serum samples were initially diluted 1:10, added to the first column of the plate and subsequently diluted 1:5 in diluent for a 12-point dilution series. After incubation and washing, plates were treated with HRP-conjugated anti-mouse total IgG, IgM or IgA and incubated for one hour at room temperature in the dark. Plates were then washed and TMB Sureblue Peroxidase Substrate was added. Once the blue color change appeared to have reached a saturation point, the reaction was quenched by adding 25 µl of 1N H2SO4 simultaneously to each well of the plate. Absorbances were read at 450nm and 570nm and Optical Density values are calculated using 450nm-570nm. Control (untreated serum) samples were used to set cutoffs and determine endpoint titers (EPTs). We observed that both traditional ELISA coating buffer and phage buffer coating resulted in the same EPTs and either can be used confidently in this assay. For total IgG responses against Muddy and Fionnbharth cohorts, we observed a time and dose dependent increase in anti-phage EPTs in serum from matched i.v. treated animals where peak responses were recorded at day 28. In addition, we did not observe measurable EPT responses to the opposite phage, suggesting these responses are phage-specific. We confirmed a very low anti-phage IgA response in the serum as expected. The serum anti-phage IgM responses were non-discernable due to high background and this suite of assays requires further optimization before deploying. In summary, we have successfully developed a reproducible and sensitive ELISA for the detection of anti-Muddy or anti-Fionnbharth mouse total IgG and IgA responses from serum samples. This methodology required no adaptation between phages for use and serves as a platform to further test anti-phage humoral immunity in the mouse model in the context of repeated deliveries and different routes of delivery for treatment of tuberculosis.