Background: Ventral midbrain (VM) dopaminergic progenitor cells derived from human pluripotent stem cells have the potential to replace endogenously lost dopamine neurons and are currently in preclinical and clinical development for treatment of Parkinson’s Disease (PD). However, one main challenge in the quality control of the cells is that rostral and caudal VM progenitors are extremely similar transcriptionally though only the caudal VM cells give rise to dopaminergic (DA) neurons with functionality relevant for cell replacement in PD. Therefore, it is critical to develop assays which can rapidly and reliably discriminate rostral from caudal VM cells during clinical manufacturing. Methods: We performed shotgun proteomics on cell culture supernatants from rostral and caudal VM progenitor cells to search for novel secreted biomarkers specific to DA progenitors from the caudal VM. Key hits were validated by qRT-PCR and ELISA. Results: We identified and validated novel secreted markers enriched in caudal VM progenitor cultures (CPE, LGI1 and PDGFC), and found these markers to correlate strongly with the expression of EN1, which is a predictive marker for successful graft outcome in DA cell transplantation products. Other markers (CNTN2 and CORIN) were found to conversely be enriched in the non-dopaminergic rostral VM cultures. Key novel ELISA markers were further validated on supernatant samples from GMP-manufactured caudal VM batches. Conclusion: As a non-invasive in-process quality control test for predicting correctly patterned batches of caudal VM DA cells during clinical manufacturing, we propose a dual ELISA panel measuring LGI1/CORIN ratios around day 16 of differentiation.