Abstract: Blastocystis, a single-celled eukaryote is a relatively common parasite that is found in the environment. Upon making contact with a human or animal host, it thrives in the lower gastrointestinal tract and spreads through the host’s stool. From the 17 recorded Subtypes (STs), ST1 and ST3 are most commonly found in human stool samples. In addition, these two subtype are widespread in Southeast Asian countries. Medical practices at large still utilize old-school methods for the detection of these subtypes despite their limitations. Therefore, this study aims to develop multiplex PCR primers for a more efficient detection of Blastocystis ST1 and ST3. The primers were designed for each STs using PerlPrimer and validated in silico using Oligo analyzer. Reference sequences were acquired from the NCBI database. PCR and gel electrophoresis were carried out, whereby the specificity of the primers were tested using non-ST1 and ST3 subtypes as well as a few common non-Blastocystis pathogens. A sensitivity test was also carried out through 10-fold dilutions of the ST1 and ST3 DNA samples. The detection primers were successfully tested, yielding target band sizes of 138 and 233 bp for ST1 and ST3, respectively. Specificity tests have confirmed that the target subtypes were detected without any false positives from non-enteric, non-Blastocystis and non-ST1 and non-ST3 variants. Primer dimers and ghosting were also ruled out from the findings. The sensitivity test has confirmed a clear detection with as low as 7 ng/μL of extracted ST1 DNA and 70 ng/μL for ST3. The multiplex PCR primers developed in this study has the potential to reduce the turnaround time, cost and labor in the detection of these human parasites. Furthermore, its use can be extended to detection of possible sources of infection, e.g., water sources, soil and even animals such as poultry and livestock. The use of these primers can greatly aid in the containment and eradication of any ongoing outbreaks as well as the prevention of reinfection due to difficulties in identifying the root cause of an epidemic.