Stem cell transplantation remains at the centre of regenerative therapy. Recent, studies have demonstrated that the therapeutic effect of stem cells originates from paracrine reactions from cellular products (secretome) rather than direct cell differentiation. The secretome (cell-free therapy) has the potential to eliminate the various challenges stem cell therapy encounters including apoptotic, inflammation, and non-immunogenic effects. Cell-free therapy is a new emerging field with the number of clinical trials slowly increasing, however, vast research currently is required in this field regarding standardization of protocols used to obtain the secretome. The recent progression of research in secretome production and characterization has created new perceptions of cellular communication and protein dynamics. Standard methods of secretome production, reproducibility, optimization, and bulk production remain a major challenge for scientists. Precise identification of biomarkers and therapeutic targets, through bioinformatics, mass spectrometry, and cell-based systems are promising techniques addressing these limitations. Therefore, this review aims to report on the methods for secretome production, collection, and characterization. Breakthroughs in this field of research will open the doors for personalized medicine, regenerative therapies, and other biotechnological applications.