The microcystin (MC) producing P. rubescens occurs in pre-alpine lakes and may impact fishery success, bathing, and raw water quality. P. rubescens extracts, characterized via LC-MS, contained the two MC-RR variants [Asp3]MC-RR and [Asp3,Dhb7]MC-RR. The protein-phosphatase-inhibition assay (cPPIA with phosphatases 1 and 2A) in its capability to quantify [Asp3]MC-RR, [Asp3,Dhb7]MC-RR, and MC-RR was compared to HPLC-DAD and anti-Adda-ELISA. The IC50 values (PP1 and PP2A) determined for MC-LR, MC-RR, and [Asp3]MC-RR were in the same range (1.9-3.8 and 0.45-0.75 nM). A 50-fold higher concentration of [Asp3,Dhb7]MC-RR (29.8 nM) was necessary to inhibit the PP2A by 50%. The PP1-IC50 of [Asp3,Dhb7]MC-RR was 22-fold higher (56.4 nM) than those of the other MCs, suggesting that specific structural characteristics are responsible for its weaker PPI capacity. Western blots demonstrated that [Asp3,Dhb7]MC-RR does not covalently bind to PP1. [Asp3,Dhb7]MC-RR has comparable in vivo LD50 values to MC-RR, despite a far lower PP-inhibiting capacity, suggesting that toxicodynamic and toxicokinetic characteristics of [Asp3,Dhb7]MC-RR are responsible for its high in vivo toxicity. The data demonstrate that cPPIA analysis of [Asp3,Dhb7]MC-RR-containing samples prevent reliable MC determination and lead to underestimation of potential toxicity.