Mastitis is a major health concern on dairy farms and causes substantial economic losses for the industry worldwide. In cases of clinical mastitis, pathogen-based treatment decisions are most economic. At present pathogens are commonly detected using aerobic culture, requiring at least 24 to 48h to obtain results. Detection of pathogens by quantitative real-time polymerase chain reaction (qPCR) offers results within a few hours. So far commercially available kits are only suitable for laboratory use. Our goal is to develop an on-farm applicable, qPCR assay to detect pathogens in milk from quarters with clinical mastitis, enabling faster management decisions and reduce the use of antimicrobials. Samples from clinical mastitis cases submitted to Quality Milk Production Services (QMPS) were tested with 2 different qPCR assays developed by Acumen Detection LLC. Assays either detected Streptococcus species (Strep. spp.) or Staphylococcus species (Staph. spp.). A baseline qPCR signal was identified using healthy quarter milk samples (control milk, California mastitis test negative, no growth after 48-h aerobic culture). To determine assay range, control milk was spiked with a serial dilution of known colony forming units of Strep. uberis and Staph. aureus. In parallel, organisms in clinical samples were identified by MALDI TOF after 48-h aerobic culture. For qPCR, milk samples were processed using the Acu-PCR Milk Prep Kit and subsequently added to the lyophilized Acu-PCR assays for Strep. spp. (STREP) and, in a multiplex assay, Staph. aureus and other Staph. spp. (STAPH, all Acumen Detection LLC). The STAPH assay detected dilutions of Staph. aureus down to 1 × 10⁵ cfu/mL and the STREP assay detected dilutions of Strep uberis down to 2 × 10⁴ cfu/mL. Control milk did not show a signal in any of the assays tested. Acu-PCR assays were able to detect the presence of Staph. spp., Staph. aureus, Strep. dysgalactiae and Strep. uberis in clinical samples confirmed by MALDI TOF. Ongoing testing is assessing specificity and sensitivity for each assay. The results show that the Acu-PCR Milk Prep Kit and the tested Acu-PCR assays are capable of detecting their respective targets in clinical milk samples.