High quality RNA products from bacterial cells are required for the molecular study. Sample preparation to acquire the high-quality RNA especially the Gram-positive bacteria like Bacillus sp., the model organism, remains a critical burden toward the integration of full molecular downstream analyses although several methods have been proposed including conventional or kit-based protocols. Those techniques were simply developed using the cell samples at certain growth stages unless some molecular studies require RNA samples gathered under different physiological stages of growth and process conditions. Herein, we developed the simple yet effective cell-lysis technique prior to RNA extraction by modifying the commercial kit-based protocols. Bacillus subtilis TL7-3 was used as the model organism in this study. Lysozyme loading (20 mg/mL) as well as the incubation time (30 min) and temperature (37 °C) was responsible for cell lysis and increased RNA concentration in the samples. Invert mixing rather than centrifugation and vortexing prevented RNA damage during protein precipitation by absolute ethanol. This was confirmed by the RNA Integrity Number (RIN) values greater than 8.0 of all RNA extracted from both vegetative cells and endospores of B. subtilis TL7-3. Additionally, absolute ethanol is preferable to our less-than-1-h protocol for protein precipitation as indicated from the higher ratios of A260/A280 and those of A260/A230 of the RNA products than 2.0 and 2.1, respectively. From the findings mentioned above, we successfully developed the modified RNA extraction protocol applicable for the intact cells of Gram-positive bacteria like Bacillus sp. at varied physiological and morphological stages.