Biostimulants, including phytohormones, are of high agricultural importance. Thus far, several methods have been developed for phytohormone detection/determination in microalgal cultures. Most of them require expensive, state-of-the-art machinery which often has limited availability in developing, agricultural countries. The main goal of our study was to develop a cheap and straightforward method for brassinosteroid determination in microalgal cultures. We used a Klebsormidium strain whose genus members have reportedly high brassinosteroid content. Using brassinolide standard, we compared the response of four different rice cultivars in a rice lamina inclination bioassay (RLIA), and from these found the variety ‘Koshihikari’ the most suitable one. A dynamic response over a broad concentration range from 0.001 to 0.1 mg/L brassinolide concentration was observed. Attempts with commonly used mechanical methods for disrupting Klebsormidium cells resulted in only negligible brassinolide release, while methanolic extraction liberated almost all cellular brassinosteroids. To overcome the negative effect of methanol on rice lamina inclination, solid-phase extraction was applied to get rid of methanol from the assay. The estimated brassinolide concentration in Klebsormidium culture by RLIA was validated using ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS).