Pathway analysis is a powerful method for discerning differentially regulated genes and elucidating their biological importance. It allows for the identification of perturbed or aberrantly expressed genes within a biological context from extensive data sets and offers a simplistic approach for interrogating such datasets. With the growing use of microarrays and RNA-Seq data for genome wide studies is growing at an alarming rate and the use of deep sequencing is revealing elements of the genome previously uncharacterised. Through the employment of pathway analysis, mechanisms in complex diseases may be explored, and novel causatives found primarily through differentially regulated genes. Further, with the implementation of next generation sequencing (NGS) a deeper resolution may be attained, particularly in identification of isoform diversity and SNP’s. Here we look at a broad overview of pathway analysis in the human brain transcriptome and its relevance in teasing out underlying causes of complex diseases. We will outline processes in data gathering and analysis of particular diseases in which these approaches have been successful.