Sclerotinia rot poses a significant challenge to the cultivation of oilseed Brassica crops, causing the reduction of seed number, weight, and quality leading to >95% yield losses globally. Due to its substantial economic impact on agriculture, Sclerotinia sclerotiorum has been extensively studied at the molecular level and has now been adopted as a model to investigate the host-pathogen interactions. Despite various procedures and strategies described in different reports, there exists a considerable disparity in how the pathogen is cultured, maintained, and manipulated across studies. This chapter provides a comprehensive guide to the fundamental procedures of working with S. sclerotiorum. It describes step-by-step methods for its routine culturing and maintenance over artificial media, replenishing and storing laboratory stocks, and conducting detached leaf assays—a robust method used for screening the mustard cultivars. Additionally, we outline the quantification of pathogen load in planta through qRT-PCR analysis. The methods presented in the current chapter are reproducible and can be suitably applied when working with different isolates of the pathogen. Through the present compilation, we aim to address the research gaps in methods involving the pathogen that would not only benefit the new researchers entering the field but also future research endeavors on white mold.