MicroRNAs in tissues and biological fluids represent a promising class of biomarkers for the molecular diagnostics and therapy of numerous diseases, including oncological diseases. Biomarkers based on easily accessible biological fluids, primarily blood-based biomarkers, are of particular value for diagnostic and prognostic purposes. To explore the potential of microRNAs as prognostic cancer markers and targets for molecular therapy, global microRNA profiling is required, which is provided by next-generation sequencing (NGS). NGS offers high sensitivity, single nucleotide resolution, and the possibility of profiling a large number of samples in parallel. Despite the promising potential of microRNAs as biomarkers and the growing number of works in this area, the literature does not address in sufficient detail the problems associated with sample preparation methods, the specifics of library preparation for microRNA sequencing, and the difficulties of quantitative analysis. Protocols for creating libraries for microRNA sequencing present specific challenges and require selecting conditions for each type of biological sample. Here, we present in detail the preparation of libraries for microRNA sequencing from pituitary adenoma tumor tissue and blood plasma of patients with acromegaly on the Illumina platform. We discuss the difficulties and limitations of the methods and evaluate the effectiveness of sequencing plasma and brain samples. This work can serve as a guide for researchers studying the mechanisms of microRNA regulation in endocrine diseases of the pituitary gland and will also allow for the adaptation of technical procedures for various biological samples in relation to other pathologies.