Embryos obtained by in vitro fertilization are currently assessed by morphology, but displays limitations with over 70% of embryos failing to implant. In this study, we performed HPLC-MS/MS analysis on the conditioned medium obtained from 50 human embryos at the 3rd day of in vitro culture. 70 proteins were identified in the medium of 48 embryos. Validation by protein array on two pools of 11 and 9 conditioned media, showed a protein pattern overlap with HPLC-MS/MS of respectively 72% and 78%. Unsupervised hierarchical cluster analysis on protein spectra, allowed to divide embryos into 3 clusters. The first cluster selectively lacked of proteins involved in programmed cell death. The third cluster was devoid of proteins involved in cell development. Embryos taking the shortest time to develop into 5 cell morulas, featuring lower implantation rate, significantly segregated in the third cluster (P=0.047). Multiple linear regression analysis, identified 12 predictive proteins for transfer success (P < 0.05). Proteomics of embryo secretome aids in understanding embryo physiology and in improving assisted reproductive technology.