A compositional statistical framework for absolute proportion estimation between fractions in RNA sequencing data. 'FracFixR' addresses the fundamental challenge in fractionated RNA-seq experiments where library preparation and sequencing depth obscure the original proportions of RNA fractions. It reconstructs original fraction proportions using non-negative linear regression, estimates the "lost" unrecoverable fraction, corrects individual transcript frequencies, and performs differential proportion testing between conditions. Supports any RNA fractionation protocol including polysome profiling, sub-cellular localization, and RNA-protein complex isolation.