Neurodegenerative disorders (NDs) are characterized by a progressive loss of neurons leading to broad symptoms affecting cognition and motor function. Despite extensive research efforts, treatment options remain palliative, and due to increasing incidence of NDs worldwide, novel therapies are necessary. Gene therapy represents an attractive approach for tackling the complex pathology of NDs. CRISPR-mediated gene activation (CRISPRa) encompasses a growing field of biotechnological approaches with exciting implications for gene therapy through upregulation of genetic targets of interest. However, reliable models are required to assess the efficiency of sgRNAs to be used for experimental approaches. Here, we present an experimental screening assay for determining the expression activity of sgRNAs using CRISPRa in vitro. Additionally, we provide a comprehensive cloning workflow for seamless tandem assembly of up to eight sgRNA candidates in a single vector using Golden Gate cloning. This strategy allows for efficient activity assessment of single or multiple sgRNA candidates simultaneously through fluorescence quantification. We identify numerous promising sgRNA candidates, demonstrating significant increase in expression of multiple genes of interest at both mRNA- and protein level in vitro. Furthermore, we investigate the effect of autophagy response in fibril-induced alpha-synuclein aggregation cultures in vitro by upregulating the expression of Tfeb. This illustrates the reliability and multiplexing potential of our workflow and presents further development of CRISPRa for gene therapy.