This study aimed to evaluate the lysine releasing rate (LR %) from 3 rumen-protected lysine (RPL) products upon mixing with TMR diets having various moisture contents, and to determine the in situ rumen stability of these PRL products. Three PRL products commercially available in Japan (A: AjiPro-L; B: LysiGEM; C: Feedtech Bypass Lysine) were used for this assessment. L-Lys contents of product A, B and C were 39.1, 53.1 and 46.8%, respectively. Each RPL (2 g) was mixed with 200 g of corn silage-based TMRs having 3 levels of moisture contents (low, L: 17.6%; medium, M: 35.6%; high, H: 61.1%). The treated TMRs were stored in plastic bags (3 replicates) at 20°C for 0, 1, 3, 6, 12, and 24 h. TMRs without RPL were used as control. At each time point, the solubilized free Lys in the treated TMR was extracted with 1 L of water. After filtration of the extracted solutions, free Lys content in the extract was analyzed, and LR % observed for the mixed TMR was calculated. Additionally, 2 ruminally fistulated lactating cows were used to determine the in situ rumen stability. The 3 RPLs were placed separately in Nylon bags (1 g/ bag: 3 replicates, 5 × 7 cm, pore size 50 μm) and incubated in the rumen for 0 (the washed control), 6, 12, and 24 h, respectively. After drying the incubated bags and extracting Lys from residues left in bags through acid hydrolysis, Lys content was analyzed by UPLC. Data were analyzed by Mixed model ANOVA of SAS. Fixed effects in the model were the RPL product, time, and their interactions. The results showed that all RPLs exhibited an increase in the LR % over storage time for M- and H-TMRs but not for L-TMR (P < 0.01). However, RPLs by time interaction was observed (P < 0.01), the LR % was highest in the product C, followed by B, and then A (67.0%, 17.0%, and 3.0% at 24 h for M, and 62.0%, 24.0%, and 4.0% at 24 h for H, respectively). The in situ ruminal protection of all RPLs was highest in the product A, followed by B, and then C (87.3%, 51.5%, and 0.2% at 24 h, respectively; RPLs by time interaction, P < 0.01). Our results indicate that it is important to investigate the RPL stability not only in the rumen but also during incorporation in the high moisture diets.