Human leukocyte antigen (HLA) typing of in vitro fertilization (IVF) embryos, aims to establish a pregnancy that is HLA compatible with an affected sibling who requires hematopoietic stem cell transplantation (HSCT). It can be performed with or without preimplantation genetic diagnosis (PGD) for exclusion of a single-gene disorder (SGD) and it is a multistep, technically challenging procedure at every stage. Our purpose was to address the difficulties of genetic analysis by developing a fast, reliable and accurate PGD-HLA protocol, to simplify patient work-up and PGD application, while providing high flexibility for combination with any SGD. Requests included PGD-HLA for β-thalassemia (β-thal)/sickle cell disease (most common request), Diamond-Blackfan anemia (DBA), chronic granulomatous disease (CGD) and preimplantation-HLA typing only. For HLA haplotyping, we selected a panel of 26 short tandem repeats (STRs) distributed across the entire HLA locus, following PGD guidelines. When required, mutation detection was performed by both a direct and indirect approach. To support concurrent SGD exclusion and HLA typing, a one-step, single-tube, multiplex fluorescent touchdown-polymerase chain reaction (PCR) was optimized. The described touchdown-PCR was successfully applied for all PGD-HLA protocols. Eight clinical cycles were performed with a diagnosis achieved for 94.7% of amplified biopsied blastomeres. Embryo transfer took place in six cycles, with two pregnancies achieved and two healthy female infants (from a twin pregnancy) born so far. Our protocol enables HLA typing in a single PCR, reducing the risk of contamination and the cost, and providing faster results. It requires minimum optimization before clinical application, irrespective of the SGD involved, decreasing the waiting time from referral to treatment for all PGD-HLA cases.